BRPI0620505A2 - polypeptide, antibody, fusion polypeptide, composition, polynucleotide, vector, host cell, method of producing a polypeptide, library of polypeptides, methods for generating a composition, method for selecting an antigen-binding variable domain, method for selecting polypeptides , method for isolating one or more polypeptides, method for treating cancer and method for treating - Google Patents

Abstract

The invention provides antibodies or fragments of these DR5 and HER-2 antigen binders. Antibodies and / or fragments of these antigen binders consist of CDR variants which comprise a diversity of highly restricted amino acid sequences. The invention also provides these polypeptides as fusion polypeptides for heterologous polypeptides, such as at least a portion of the phage or viral capsid proteins, markers and ligands. It also provides compositions and methods of use for the treatment of cancer and related autoimmune diseases.

Description

"POLYPEPTIDES, ANTIBODY, FUSION POLYPEPTIDE, COMPOSITION, polynucleotide, VECTOR, host cell, PRODUCTION METHOD OF A POLYPEPTIDE, DEPOLIPEPTÍDEOS LIBRARY, METHODS TO GENERATE A COMPOSITION, MÉTODOPARA SELECTING A VARIABLE DOMAIN BINDING TO ANTIGEN, METHOD TO SELECT POLYPEPTIDES, METHOD FOR ISOLARUM OR MORE POLYPEPTIDES, METHOD FOR DECENT TREATMENT AND METHOD FOR TREATMENT "

This is a non-provisional application claiming priority for US Patent Serial Number 60/742185 filed December 2, 2005 and US Patent Serial Number 60/805553 filed June 22, 2006, from which all applications are incorporated herein. by reference.

Field Of Invention

The present invention relates to highly diverse CDR variants using highly restricted amino acid repertoires and libraries containing a variety of such sequences. The invention also relates to fusion polypeptides containing these CDR variants. The invention also relates to methods and compositions useful for the identification of novel binding polypeptides which may be used therapeutically or as reagents.

Background of the Invention

Phage display technology has provided a powerful tool for generating and selecting new proteins that bind to a binder, such as an antigen. The use of phage display techniques allows the generation of large libraries of protein variants that can be rapidly selected for those that bind to highly affinity target antigen sequences. Nucleic acids encoding variant polypeptides are fused to a sequence of nucleic acids that encode a viral capsid protein, such as gene III protein or gene VIII aprotein. Phagomonovalent display systems have been developed in which nucleic acid sequences encoding a protein or polypeptide are fused to a nucleic acid sequence encoding a portion of the gene III protein (Bass, S., Proteins, 8: 309 (1990)). Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3: 205 (1991)). In a monovalent phage display system, the fusion gene is expressed at low levels and wild-type gene III proteins are also expressed so that particle infectivity is maintained. Methods for generating peptide libraries and the selection of such libraries have been disclosed in many patents (e.g., US Patent 5,723,286, US Patent 5,432,018, US Patent 5,580,717, US Patent 4,427,908 and US Patent 5,498,530).

Demonstration of filamentous phage surface peptide expression and expression of E. coli noperiplasma functional antibody fragments was important in the development of phage display library antibodies Smith et al., Science (1985), 228: 1315; Skerra and Pluckthun , Science (1988), 240: 1038). Libraries of antibodies or polypeptide-binding antigens have been prepared in a number of ways, including altering a single gene, randomly inserting DNA sequences, or cloning a family of related genes. Methods for the presentation of antibody-binding or antigen-binding fragments using phage display have been described in U.S. Patent Nos. 5,750,373, US5733743, US 5837242, US 5969108, US 6172197, US 5580717 and US 5658727.

The library is then selected for expression of antibodies or protein-binding antigens with the desired characteristics.

Phage display technology has several advantages over conventional hybridoma technology and recombinant antibody preparation methods with the desired characteristics. This technology allows the development of large antibody libraries with diverse sequences in less time and without the use of animals. The preparation of humanized hybridomas or antibodies can easily require several months of preparation. In addition, since immunization is not required, phage library antibodies can be generated by toxic or low antigenicity antigens (Hogenboom, Immunotechniques (1988), 4: 1-20). Phage antibody libraries can also be used to pause and identify new human antibodies.

Antibodies have become very useful as therapeutic agents for a wide variety of conditions. For example, humanized antibodies against HER-2, a tumor antigen, useful in the diagnosis and treatment of cancer. Other antibodies, such as anti-INF-γ antibodies, are useful in treating inflammatory conditions such as Crohn's disease. Phage display libraries have been used to generate human antibodies from immunized and non-immunized humans, precursor sequences, cells Ig naives and repertoires (Barbas & Burton, Trends Biotech (1996), 14: 230; Griffiths et al., EMBO J. (1994), 13: 3245; Vaughan et al. Nat. Biotech. (1996), 14: 309; Winter patent EP 0368,684 BI). Libraries of naive or non-immune ligand antigens were generated using a variety of lymphoid tissues. Some of these libraries are commercially available, such as those developed by Cambridge Antibody Technology and Morphosys (Vaughan et al., Nature Biotech 14: 309 (1996); Knappik et al., J. Mol. Bioi 296: 57 (1999)). However, many of these libraries have limited diversity.

The ability to identify and isolate high affinity antibodies in a phage display library is important for identifying novel human antibodies for therapeutic purposes. Isolation of high affinity antibodies in a library is traditionally associated with being dependent, at least in part, on the size of the library, the efficiency of bacterial cell production and the diversity of the library. See, for example, Knappik et al., J. Moi Biol. (1999), 296: 57. The size of the library is decreased by inefficiency of production due to misfolding of antibody protein or binding antigen and the presence of codons encountered. Expression in bacterial cells may be inhibited if the antibody or binding antigen domain is not properly folded. Expression may be enhanced by mutations in residues and modifications of the inter-variable / constant, or in selected CDR residues. (Deng et al., J. Biol. Chem. (1994), 269: 9533, Ulrich et al., PNAS (1995), 92: 11907-11911; Forsbergetal., J. Biol. Chem. (1997), 272: 12430). ). The sequence of the structural region is a factor in providing adequate folding when phage antibody libraries are produced in bacterial cells.

Generating a diverse library of antibody or ligand antigen proteins is also important for isolating high affinity antibodies. Libraries with limited CDR diversifications have been generated using a variety of approaches. See, for example, Tomlinson, Nature Biotech. (2000), 18: 989-994. The CDR3 regions are of interest, in part because they are often antigen-binding participants. The heavy chain CDR3 regions vary greatly in size, sequence and structural conformation.

Others have also generated diversity by randomizing light and heavy chain variable CDR regions using all 20 amino acids from each position. Using all 20 amino acids was thought to result in a great diversity of variant antibody sequences and increase the chance of identifying new antibodies. (Barbas, PNAS., 91: 3809 (1994); Yelton, DE, J. Immunology, 155: 1994 (1995); Jackson, JR, J. Immunology, 154: 3310 (1995) and Hawkins, RE, J. Mol. Biology, 226: 889 (1992)).

Attempts are also made to create diversity by restricting the amino acid substitution group on some CDRs to reflect amino acid distribution in naturally occurring antibodies. See, Garrard & Henner, Gene (1993), 128: 103; Knappik et al., J. Mol. Biol. (1999), 296: 57. However, these attempts have had varying success and have not been systematically and quantitatively applied. Creating diversity in the CDR regions while minimizing the number of amino acid changes has been a challenge. Also, in some cases, since a first library was generated according to a set of criteria, it may be desirable to further increase the diversity of the first library. However, this requires that the first library have sufficient diversity and yet remain sufficiently small in size as well as greater diversity can be introduced without substantially higher limitations such as yield, etc.

Some groups have reported in theoretical and experimental analyzes of the minimum number of amino acid repertoire that is required for protein generation. However, these analyzes were generally limited in scope and nature, and questions remained regarding the feasibility and applicability of complex-function polypeptide production using a restricted set of amino acid types. See, for example, Riddle and he, Nat. Struct. Biol. (1997), 4 (10): 805-809; Shang et al., Proc. Natl. Acad. Know. USA (1994), 91: 8373-8377; Heinz et al., Proc. Natl Acad.Sei. USA (1992), 89: 3751-3755; Regan & Degradated, Science (1988), 241: 976-978; Kamteker et al., Science (1993), 262: 1680-1685; Wang & Wang, NatStruct. Biol. (1999), 6 (11): 1033-1038; Xiong et al., Proc. Natl. Acad. Know. USA (1995), 92: 6349-6353; Heinz et al., Proc. Natl. Acad. Know. USA (1992), 89: 3751-3755; Cannata et al., Bioinformatics (2002), 18 (8): 1102-1108; Davidson hey, Nat. Struct. Biol. (1995), 2 (10): 856-863; Murphy et al., Prot. Eng. (2000), 13 (3): 149-152; Brown & Sauer, Proc. Nati Acad. Know. USA (1999), 96: 1983-1988; Akanuma hey there, Proc. Nati Acad. Know. (2002), 99 (21): 13549-13553; Chan, Nat. Struct. Biol. (1999), 6 (11): 994-996.

Thus, there remains a need to improve library generation methods that have a sufficient degree of diversity, are still sufficiently condescending for further manipulations directed at further diversification of the expression of distress, etc. The present invention described satisfies this need and provides other benefits.

Brief Description of the Invention

The present invention provides simplified and flexible methods for producing polypeptides comprising variant CDRs consisting of restricted diversity sequences that still retain the ability to bind to the target antigen. Contrary to conventional methods based on the proposition that adequate diversity of the target antigen binders can be generated only if a given CDR (s), all CDRs are diversified and, unlike conventional notions that adequate diversity is dependent on A wide range of amino acid substitutions (generally by replacing all or most of the 20 amino acids), the invention provides methods capable of generating high quality target-binding polypeptides that are not necessarily dependent on a particular diversification of CDR (s) or a certain number of amino acids. CDRs of a reference polypeptide or source antibody. The invention is based, at least in part, on the surprising and unexpected finding that highly diverse functional polypeptide libraries that include high quality antigen-binding can be generated by a minimally diversified number of amino acid depositions with a very restricted number of. amino acid residues. The methods of the invention are fast, convenient and flexible, based on the use of a restricted codon set encoding a small number of amino acids. The diversity of the restricted sequence, and therefore generally smaller population size (e.g. libraries) of polypeptides generated by the methods of the invention, allows for greater diversity of these populations, whenever necessary or desired. This is an advantage not generally provided by conventional methods.

Candidate binding polypeptides generated by the invention have high quality antigen binding characteristics and have structural characteristics which provide for high yield of cell culture production. The invention provides methods for generating such binding polypeptides, methods of using such polypeptides and compositions containing them.

In one aspect, the invention provides fusion polypeptides comprising diverse CDR (s) and a heterologous polypeptide sequence (in certain embodiments having at least a portion of a viral polypeptide), as single polypeptide and as a member of a single polypeptide plurality which are binder candidates to targets of interest. Compositions (such as libraries) comprising such polypeptides find use in a variety of applications, for example, as pooling candidate immunoglobulin polypeptides (e.g., antibodies and antibody fragments) that bind to targets of interest. Such polypeptides may also be generated using non-immunoglobulin scaffolds (e.g., proteins such as human growth hormone and the like). The invention encompasses various aspects, including polynucleotides and polypeptides produced according to the methods of the invention and of systems, kits and articles of manufacture for practicing the methods of the invention, and / or use of the polypeptides / polynucleotides and / or compositions of the invention.

In one aspect, the invention provides a method for generating a polypeptide including at least one, two, three, four, five or all variant CDRs selected from the group consisting of H1, H2, H3, L1, L2 and L3, wherein said polypeptide is capable of of binding to a target antigen of interest, said method comprises identifying at least one (or any number of all) accessible solvent and the highly diverse amino acid position in a CDR reference corresponding to the CDR variant, and; (ii) varying the amino acid in the accessible solvent and highly diversified position, generating several exemplary variant CDRs using a restricted codon set (the definition of "restricted codon set" is specified below).

Several aspects and examples of embodiments of the invention methods are useful for the production and / or use of a pool containing a plurality of polypeptides of the invention, in particular for the selection and identification of candidate ligands to target antigen of interest. For example, the invention provides a method for generating a composition comprising a plurality of polypeptides, each polypeptide including at least one, two, three, four, five or all CDR variants selected from the group of CDRs consisting of H1, H2, H3, L1. , L2 and L3, where these polypeptides are capable of binding to a target antigen of interest, the diode method comprises identifying at least one (or any number up to the accessible) solvent and highly diverse amino acid position in a CDR reference corresponding to the variant. CDR; and (ii) varying the amino acid in the accessible solvent and the highly diverse position, generating several exemplary CDR variants using a codon restricted set; wherein a plurality of polypeptides are generated by amplifying a template polynucleotide with a set of oligonucleotides consisting of a highly restrictive degeneracy in the coding sequence of a variant amino acid, wherein said highly restrictive degeneracy reflects the limited number of codon sequences of the restricted codon set.

In another example, the invention provides a method comprising constructing an expression vector containing a polynucleotide sequence encoding a light chain, a heavy chain, or both light chain and heavy chain variable domains of a source antibody including at least one, two, three, four, five or all of the CDRs selected from the CDR group consisting of L1, L2, L3, H1, H2, H3 and mutating at least one, two, three, four, five or all CDRs of the source antibody by at least one ( or any number to all) accessible solvent and highly diverse amino acid position using a restricted codon set.

In another example, the invention provides a method that includes: constructing a phage library or phagemid particles exhibiting a plurality of polypeptides of the invention; exposing the library to departments with a target antigen under conditions suitable for binding the particles to the target antigen; and separating the binding particles from those not binding to the antigen.

In one of the methods of the invention described herein, an accessible solvent and / or highly diverse amino acid position may be any position meeting the criteria as described herein, particularly any combination of positions as described herein, for example, any combination of polypeptide positions. described for the invention (as described in more detail herein). Suitable amino acid variants may be any that meet the criteria as described herein, for example, variant amino acids in the polypeptides of the invention, as described in more detail below. Development of CDR variety varieties may involve development of diversity in length and / or sequence. of the CDR.

For example, CDRH3 may be diversified in length, for example, 7 to 21 amino acids in length, and / or in sequence, for example, highly diverse variability and / or solvent position accessible with amino acids encoded by a restricted codon set. . In some embodiments, a portion of the CDRH3 has a length ranging from 5 to 21, 7 to 20, 9 to 15, or 11 to 13 amino acids, and has an amino acid variation at one or more positions encoded by a restricted codon set that encodes a limited number. amino acids such as the restricted codon set encoding no more than 19, 15, 10, 8, 6, 4 or 2 amino acids. In some examples, the C-terminal end has an amino acid sequence of AM, AMDY, or DY.

In some embodiments, the polypeptides of the invention may be in a variety of forms as long as the polypeptide binding function is maintained. In some embodiments, a polypeptide of the invention is a fusion polypeptide (i.e., a fusion of two or more heterologous polypeptide sequences). Common CDR polypeptides according to the invention may be prepared as a fusion of polypeptides of at least a portion of a viral capsid protein, for example for use in phage exposure. Viral capsid proteins that may be used for the display of the polypeptides of the invention comprise p III proteins, pVIII main phage protein, Soc (T4 phage), Hoc (T4 phage), gpD (phage lambda), pVI, or variants or fragments thereof. In some embodiments, the polypeptide is fused to at least a portion of a viral capsid protein, such as a virally selected capsid protein from the group consisting of, p11, pVIII, Soc, Hoc, gpD, pVI, evariates, or fragments thereof. In some embodiments, wherein the diverse CDRs polypeptide is one or more antibody variable domains, the antibody variable domain may be displayed on the surface of the virus in a variety of ways, including ScFv, Fab, ScFv2, F (ab ') 2. and F (ab) 2-. For bivalently exposing polypeptides, the fusion protein in certain instances includes a dimerization domain. The dimerization domain may include a dimerization sequence and / or a sequence that includes one or more residuoscistein. The dimerization domain may be linked, directly or indirectly, to the C-terminal end of a heavy chain or domain-constant variable domain (e.g., CH1). The structure of the dimerization domain may serve, depending on whether the antibody variable domain is produced as a fusion protein component with the capsideviral protein component (for example, without a stop codon after the dimerization domain) or if the domain The antibody variable is produced predominantly without the viral capsid protein component (for example, with a codon encountered after the dimerization domain). When the antibody variable domain is produced predominantly as a component protein fusion protein of the viral capsid, one or more disulfide bonds or single dimerization sequence provides a bivalent display. For predominantly produced antibody variable domains not fused to a component of the viral capsid protein (e.g., with an amber stop codon), the dimerization domain may include both a cysteine residue and a dimerization sequence.

In addition, optionally, a fusion polypeptide may include a tag that may be useful for purification, detection and / or screening, such as FLAG, poly-his, gD tag, c-myc, fluorescent protein or β-galactosidase. In one example, a fusion polypeptide comprises fusion of a light chain constant or variable domain to a labeled polypeptide. In another aspect of the invention, a polypeptide, such as an antibody variable domain, is obtained from a single source or Molecule Mold. The source or template molecule may be selected or developed for characteristics such as good yield and stability when producing eukaryotic or prokaryotic cell culture and / or to accommodate CDR H3 regions of varying dimensions. The sequence of the template molecule may be altered to improve folding and / or variable domain display when presented as a fusion protein with a phagesid phage protein. For example, a source antibody may comprise the variable domain amino acid sequence of the humanized antibody 4D5 (light chain variable domain (Figure 1; SEQ ID No: 1)); (heavy chain variable domain (Figure 1; SEQ ID No: 2)). For example, in a light or heavy chain antibody variable domain, framework region residues may be modified or altered from the source antibody or model molecule to improve folding, yield, presentation or affinity of the antibody variable domain. In some embodiments, framework region residues are selected to be modified from the source antibody or template molecule when the amino acid in the structural region position of the source molecule is different from the amino acid or amino acids commonly found at the position in which they naturally occur in the antibodies or in a subgroup. of the consensus sequence. Amino acids at these positions may be altered by the amino acids most commonly found in naturally occurring antibodies or in a subset of the consensus sequence at that position. In one example, the heavy chain structural region 71 residue may be R, V or A. In another example, the heavy chain structural region residue 93 may be S or A. In still another example, the structural region residue 94 is R, K or T or is coded by MRT. In another example, a frame residue 93 and 94 frame residue is still R. In another example, the heavy chain framework49 residue may be alanine or glycine. Residues of the light chain structural region may also be altered. For example, the amino acid at position 66 could be arginine or glycine. The structural region for the light and heavy chain sequence of the wild type humanized 4D5 antibody are shown in Figure 6 (SEQ ID Nos: 6-9 and 10-13, respectively). The structural regions for the heavy chain sequence of the humanized antibody 4D5 in which the light chain is modified at position 66 and the heavy chain is modified at positions 71, 73, and 78 are shown in Figure 7 (SEQ ID Nos: 14-17 and 18-21, respectively).

The methods of the invention are capable of generating a large variety of polypeptides comprising a diverse set of CDR sequences. In one example, one or more libraries are formed using the methods of the invention as described herein. The Libraries are selected for binding to target antigen, eg human DR5 and HER-2.

The randomized daimmunoglobulin heavy chain variable domain is provided to provide diversity. In one embodiment, a polypeptide comprises an immunoglobulin heavy chain variable domain, where:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein G is position 26 and X1 is position 28 according to the system Kabat numbering; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Ye S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X.14-X15-X16-DY (SEQ ID NO: ED 31), wherein X1 is position 95 according to the Kabat numbering system, and wherein X1 is selected from R, Y, M; X 2 is selected from Y and R; X3 is selected from Y, S, R, P and G; X4 is selected from Y and S; X5 is selected from Y, S, R and H; X6 is selected from R, Y and S; X7 is selected from G, Y and S; X8 is selected from R, Y and S; X9 is selected from G, Y and S; X10 is selected from R, Ye S; X11 is selected from G, Y and S; X 12 is selected from S, Y, R, G and A; X13 is selected from G and Y; X 14 is selected from L, M, R, G and A; and X15 is selected from G, F and L or is not present, and X16 is F or not present.

In another example, a polypeptide comprises an immunoglobulin heavy chain variable domain, where:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein G is position 26 and X1 is position 28 according to the system Kabat numbering; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Ye S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 24), wherein X1 is position 95 according to the Kabat numbering system, characterized in that the amino acids in each X1-X6 deposition are selected from an amino acid pool at a 50 molar moiety. % Y, 25% S, and 25% G; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 50% Y, 25% S and 25% G, whether present or not; where X18 is selected from G and A, and where X19 is selected from I, M, L and F.

In another example, a polypeptide comprises an immunoglobulin heavy chain variable domain, where:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein G is position 26 and X1 is position 28 according to the system Kabat numbering; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50, according to Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ ID No: 26), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio of 25% Y, 50% S, and 25% R; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 25% Y, 50% S and 25% R, or not present; wherein X18 is selected from Ge A; and wherein, X19 is selected from I, M, L and F.

In another example, a polypeptide comprises an immunoglobulin heavy chain variable domain, where:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein G is position 26 and X1 is position 28 according to the system Kabat numbering; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Ye S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X 14-X 15-X 16-X 17-X 18 -X 19-DY (SEQ ID NO: 27), where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from a amino acid pool in a molar ratio of 38% Y, 25% S, and 25% G and 12% R, characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 38% Y, 25% S, and 25% G and 12% R, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, Le F.

In another example, a polypeptide comprises an immunoglobulin heavy chain variable domain, where:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein G is position 26 and X1 is position 28 according to the system Kabat numbering; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Ye S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( In which: X1 is position 95 according to the Kabat numbering system, characterized in that the amino acids in each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V and 1% W, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F.

In one aspect of the invention, CDRH1 comprises at least one amino acid sequence selected from any of IDN SEQs: 524-540 and 189-294 or at least one amino acid sequence selected from any of the sequences in Figure 11A or Figure 15. ACDRH2 comprises at least one selected amino acid sequence of any of SEQ ID Nos: .541-557 and 295-400 or at least the amino acid sequence selected from either sequence in Figure 11A or Figure 15. CDRH3 comprises at least one sequence amino acid sequence selected from any of IDN SEQs: .558-574 and 401-506 or at least the selected CDRH3 amino acid sequence from any of the sequences in Figure 11A or Figure 15.

In another aspect of the invention, the amino acid sequence CDRH3 comprises X1 is selected from R and Y; X3 is S; X8 is S; X9 is Y and X10 is Y or R. In another aspect of the invention, the CDRH3 amino acid sequence comprises the amino acid sequence X1-R-S-Y-R-Y-G-Y-X10-G-S-Y-X14-F-D-Y (SEQ ID NO: 575).

In another aspect of the invention, the polypeptide binds to human DR5 or binds human DR5 and murine DR5. In one aspect of the invention the opolipeptide is an antibody. In one embodiment, the antibody contains a heavy chain variable domain comprising: i) a CDRH1 containing a GFYISSSSIH amino acid sequence (SEQ ID NO: 576); ii) a CDRH2 containing an amino acid sequence SISPSSGSTYYADSVKG (SEQ NO ID: 577); and iii) a CDRH3 containing an amino acid sequence YRSYRYGSYYGSYGFDY (SEQ NO ID: 578). In one embodiment, the antibody contains a heavy chain variable domain comprising: i) a CDRH1 containing a GFYISSSSIH amino acid sequence (SEQ ID No: 579); ii) a CDRH2 containing an amino acid sequence SISPSSGYTSYADSVKG (SEQ NO ID: 580); and iii) a CDRH3 containing an amino acid sequenceRRSYRYGSYRGSYAFDY (SEQ NO ID: 581).

In another aspect, the polypeptide further comprises a light chain variable domain in which:

(i) CDRL3 comprises an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: 25); where X1 is position 91 and is selected from Y, H and S; X 2 is selected from Y and S; X3 is selected from Y, S and T, X4 is selected from Y, S and T, and X5 is selected from S, P and Y. In one embodiment, a CDRL1 having the amino acid sequence RASDVNTAVA (SEQ ID NO: 29). In one embodiment, a CDRL2 having the SASSLYS amino acid sequence (SEQ ID NO: 30).

In one embodiment, the antibody contains a heavy chain variable domain comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein, X1 is apposition 28 according to the Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y, X5 is selected from S and Y, and X6 is selected from S and Y; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-DY (SEQ ID NO: 582), wherein X1 is the position of amino acid 95 according to the numbering system of Kabat e is selected from S and Y; X2 is selected from S and Y and R; X3 is selected from S, G, Y and H; X4 is selected from S, G, Y and R; X5 is selected from G and A; X6 is selected from F, M, L and A; and X7 is selected from F, M and L or is missing.

In one aspect of the invention, the CDRH1 comprises at least one amino acid sequence selected from any of IDN SEQs: 189-198. CDRH2 comprises at least one selected amino acid sequence from any of SEQ ID Nos: 295-304. ACDRH3 comprises at least one selected amino acid sequence of any of SEQ ID Nos: 401-410.

In another embodiment, the antibody contains a heavy chain variable domain comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein, X1 is apposition 28 according to the Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y, X5 is selected from S and Y, and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-DY (SEQ ID NO: 583) wherein X1 is the position of amino acid 95 according to the Kabate numbering system is selected from Y, S and G; X 2 is selected from Y, S, G, R and A; X3 is selected from G, Y, S and R; X4 is selected from G, Y and F; X5 is selected from Y, S, N and G; X6 is selected from Y, R, H and W; and X7 is selected from G and A; and X8 is selected from F, M, L and I.

In one aspect of the invention, the CDRH1 comprises at least one amino acid sequence selected from any of SEQ IDNs: 199-216. CDRH2 comprises at least one selected amino acid sequence from any of SEQ ID Nos: 305-322. ACDRH3 comprises at least one selected amino acid sequence of any of SEQ ID Nos: 411-428.

In another embodiment, the antibody contains a heavy chain variable domain comprising:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein X1 is position 28 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat's numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y, and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 584), wherein X1 is the position of amino acid 95 is selected from Y, S, R and G; X 2 is selected from Y, S, R and G; X3 is selected from S, Y, G and W; X4 is selected from S, Y, G and Q; X5 is selected from G, Y and S; X6 is selected from G, Y S, R and V; X 7 is selected from S, Y, G and R; X8 is selected from Y, S, G, R, P and V. X9 is selected from G, A, Y, S and R; X10 is selected from M, F, G, Y, S and R X11 is selected from A, Y, S, G and R; X 12 is selected from I, M, F, L, A, G, S, Y, Re T; X13 is selected from F, M, L, G, A, Y, T and S or is not present; X14 is selected from L, M, F, I, G, Y, A and T; and X15 is selected from M, L, Y, G and R or is not present; X16 is selected from Y and G or is not present; X17 is selected from R, M and G or is not present; X18 is selected from P and Aou is not present; and X18 is L or not present.

In another embodiment, a polypeptide comprises an immunoglobulin heavy chain variable domain, in which:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), characterized in that G is position 26 and X1 is position 28 according to with the Kabat numbering system; and wherein X1-X5 are naturally present amino acids other than cysteine;

(ii) CDRH2 comprises an amino acid sequence: X6-I-X7-P-X8-X9-G-X10-T-X11-YADSVKG, where X6 is position 50 according to the Kabat numbering system, and wherein X6-X11 are naturally present amino acids other than cysteine; and

(iii) CDRH3 comprises an amino acid sequence: X12-X13-X14-X15-X16- (X17) n-X18-X19-D-Y, wherein X12 is position 95 according to the Kabat numbering system; and wherein n is an appropriate number which will maintain the functional activity of the heavy chain variable domain; and noqual, X12-X19 are naturally present amino acids other than cysteine. In one aspect, n is a number from 1 to 12. In one aspect, X1 is selected from Y and S; X2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S. In one aspect, X6 is selected from Y and S; X7 is selected from Y and S; X8 is selected from Y and S; X9 is selected from Y and S; X10 is selected from Y and S; eX11 is selected from Y and S. In one aspect, the amino acids at each of the X12-X17 positions are selected from an amino acid pool at a molar ratio of 50% Y, 25% S and 25% G, and X18 is selected from G and A, and X19 is selected from I, M, L, and F. In an alternative aspect, amino acids at each of the positions X12-X17 are selected from an amino acid pool at a ratio molar of 25% Y, 50% S and 25% R, where X18 is selected from G and A, and X19 is selected from I, M, L, and F. In an alternative aspect, the amino acids at each position X12-X17 are selected from an amino acid pool at a molar ratio of 38% Y, 25% S, 25% G and 12% R, where X18 is selected from G and A , and X19 is selected from I, M, L, and F. In another alternative aspect, amino acids at each of the positions X12-X17 are selected from an amino acid pool at a ratio m 20% Y olar, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; where X18 is selected from G and A; and X19 is selected from I, M, L, and F. In one aspect of the invention, CDRH1 comprises at least one amino acid sequence selected from any of SEQ ID Nos: 217-294 or one of the CDRH1 sequences in Figure 11. A CDRH2 comprises at least one amino acid sequence selected from any of SEQ ID Nos: 323-400 or one of the CDRH2 sequences in Figure 11. CDRH3 comprises at least one amino acid sequence selected from any of IDN SEQs: 429-506 or one CDRH3 sequences in Figure 11.

In another embodiment, a polypeptide comprising an immunoglobulin heavy chain variable domain is provided, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No. : _), characterized by the fact that G is apposition 26 and X1 is position 28 according to the deKabat numbering system; wherein X1-X5 are naturally present amino acids other than cysteine;

(ii) CDRH2 comprises an amino acid sequence: X6-I-X7-P-X8-X9-S-X10-T-X11-YADSVKG (SEQ ID No.:_), wherein X6 is apposition 50 according to the Kabat numbering system, and in which X6-X11 are naturally present amino acids other than cysteine; and

(iii) CDRH3 comprises an amino acid sequence: X12-X13-X14- (X15) n-X16-X17 (SEQ ID No.:_), wherein X14 is position 95 according to the Kabat numbering system; and wherein, n is an adequate number that will maintain the functional activity of the heavy chain variable domain; and wherein, X12-X17 are naturally present amino acids other than cysteine. In one aspect, n is a number from 1 to 14. In another aspect, X1 is selected from Y and S; X2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; and X6 is selected from Y and S. In another aspect, X1 is selected from W and S; X2 is selected from W and S; X3 is selected from W and S; X4 is selected from W and S and X5 is selected from W and S. In another aspect, X1 is selected from R and S; X 2 is selected from R and S; X3 is selected from R and S X4 is selected from R and S and X5 is selected from R and S. In another aspect, X1 is selected from F and S; X 2 is selected from F and S; X3 is selected from F and S; X4 is selected from F and S and X5 is selected from F and S. In another aspect, X6 is selected from Y and S; X7 is selected from Y and S; X8 is selected from Y and S; X9 is selected from Y and S; X10 is selected from Y and S; eX11 is selected from Y and S. In another aspect, X6 is selected from W and S; X7 is selected from W and S; X8 is selected from W and S; X9 is selected from W and S; X10 is selected from We S; and X11 is selected from W and S. In another aspect, X6 is selected from R and S; X7 is selected from R and S; X8 is selected from R and S; X9 is selected from R and S; X10 is selected from R and S; and X11 is selected from R and S. In another aspect, X6 is selected from F and S; X7 is selected from F and S. X8 is selected from F and S; X9 is selected from F and S; X10 is selected from F and S; and X11 is selected from F and S. In another aspect, X12 is selected from Y and S; X13 is selected from Y and S; X14 is selected from Y and S; X15 is selected from Y and S. X16 is selected from G and A; and X17 is selected from F, L, I and M.

In another aspect, X12 is selected from W and S; X13 is selected from W and S; X14 is selected from W and S; X15 is selected from W and S; X16 is selected from G and A; and X 17 is selected from F, L, I and M. In another aspect, X 12 is selected from R and S; X13 is selected from R and S; X14 is selected from R and S; X15 is selected from R and S; X16 is selected from G and A; and X17 is selected from F, L, I and M. In another aspect, X12 is selected from F and S; X13 is selected from F and S; X14 is selected from F and S; X15 is selected from F and S; X16 is selected from Ge A; and X17 is selected from F, L, I and M.

In another aspect, the amino acids at each of the positions of X1-X15 are selected from S and one from A, C, F, G, I, L, N, P, R, T, W or Y, X16 is selected. from G and A; and X17 is selected from F, L, I, and M.

In another embodiment, an immunoglobulin heavy chain variable domain polypeptide, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH, characterized in that where G is apposition 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-Y-A-D-S-V-K-G, characterized by the fact that X1 is position 50 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S, where X4 is selected from Y and S; where X5 is selected from Y and S; and wherein, X 6 is selected from Y and S; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 fact that X1 is apposition 95 according to the Kabat numbering system, and in which the amino acids in each of the X1-X17 depositions are selected from A, C, F, G, I, L, N, P, R, T, W or Y, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M.

In one aspect of the invention, the CDRH1 comprises at least amino acid sequence selected from either IDN SEQs: 816-842 or one of the CDRH1 sequences in Figure 21A. CDRH2 comprises at least one amino acid sequence selected from any of SEQ ID Nos: 843-869 or one of the CDRH2 sequences in Figure 21A. CDRH3 comprises at least one selected amino acid sequence from any of SEQ ID Nos: 870-896 or one of the CDRH3 sequences in Figure 21A.

In another example, a polypeptide containing an immunoglobulin heavy chain variable domain, in which:

(i) CDRH1 comprises an amino acid sequence: G-F-X1-I-X2-X3-X4-X5-I-H, characterized in that G is position 26 and X1 is

position 28 according to the Kabat numbering system; and wherein the amino acids at each of the positions of X1-X5 are selected from S and one of Y, W, R or F; and

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is apposition 50 according to the Kabat numbering system ; and wherein the amino acids at each of the positions of X1-X6 are selected from S and one of Y, W, R or F; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 fact that X1 is position 95 according to the Kabat numbering system, and the amino acids at each of the positions of X1-X19 are selected from S and one of Y, W, R or F, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M.

In one aspect of the invention, the CDRH1 comprises at least one amino acid sequence selected from either IDN SEQs: 924-950 or one of the CDRH1 sequences in Figure 24A. CDRH2 comprises at least one amino acid sequence selected from any of SEQ ID Nos: 951-977 or one of the CDRH2 sequences in Figure 24A. CDRH3 comprises at least one selected amino acid sequence from either of SEQ ID Nos: 978-1004 or one of the CDRH3 sequences in Figure 24A.

In one aspect of the invention, the polypeptide binds to human HER-2. In a further aspect of the invention, the polypeptide comprises an antibody. In one aspect of the invention, the antibody comprises a heavy chain variable domain comprising;

i) a CDRH1 containing an amino acid sequence GFSIYSSYIH (SEQ ID No: 821);

ii) a CDRH2 containing an amino acid sequenceSIYPYSGYTSYADSVKG (SEQ ID No: 848); and

iii) a CDRH3 containing an amino acid sequence WWSSAFDY (SEQ ID No: 875). In another aspect of the invention, the antibody comprises a heavy chain variable domain comprising;

i) a CDRH1 containing an amino acid sequence GFSIWWSWIH (SEQ ID NO: 932);

ii) a CDRH2 containing an amino acid sequenceSISPSSGWTSYADSVKG (SEQ ID No: 959); and

iii) a CDRH3 containing a WWSSAMDY amino acid sequence (SEQ ID No: 986). In another aspect of the invention, the antibody comprises a heavy chain variable domain comprising;

i) a CDRH1 containing an amino acid sequence GFSISSSYIH (SEQ ID No: 944);

ii) a CDRH2 containing an amino acid sequenceSIYPYSGYTSYADSVKG (SEQ ID No: 971); and

iii) a CDRH3 containing a YYSYALDY amino acid sequence (SEQ ID No: 998). In another aspect of the invention, the antibody comprises a heavy chain variable domain comprising;

i) a CDRH1 containing a GFYISSSSIH amino acid sequence (SEQ ID No: 230);

ii) a CDRH2 containing an amino acid sequence YIYPSSGYTSYADSVKG (SEQ ID No: 336); and

iii) a CDRH3 containing a GYYYSYYSGYALDY amino acid sequence (SEQ ID No: 442).

In another aspect of the invention, the antibody is comprised of a light chain variable domain in which CDRL3 comprises an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (IDSEQ NO: 25); characterized in that X1 is position 91 according to the Kabat numbering system and is selected from S and Y; X2 is selected from S, Y and F; X3 is selected from Y, S and F; X4 is selected from Y and S, and X5 is selected from S and Y.

In another aspect of the invention, an antibody is provided which comprises a light chain variable domain having a CDRL3 sequence having the amino acid sequence QQ-X1-X2-X3-X4-P-X5-T (SEQ ID NO: _), in which X1 is position 91 according to the numbering of Kabat and is selected from Y and S; X2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from S and Y. In another aspect of the invention, an antibody comprising a light chain variable domain having a CDRL3 sequence having the amino acid sequence QQ-X1-X2-X3-X4-P-X5-T is provided. wherein X1 is position 91 according to the Kabat numbering and the amino acids at each of the X1-X5 positions are selected from. If one of W, R or F. In one aspect of the invention, CDRL3 comprises at least an amino acid sequence selected from any of SEQ ID Nos: 83-188, 507-523, 789-815 and 879-923 or one of the CDRL3 sequences in Figure 11, 15, 21 or 24.

In another aspect of the invention there is provided a polypeptide comprising an immunoglobulin light chain variable domain in which:

(i) CDRL1 comprises a first hypervariable consensus sequence or variant thereof containing a substitution in one or more positions as compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprises a second hypervariable consensus sequence or variant thereof containing a substitution in one or more positions, as compared to the corresponding hypervariable consensus sequence, and

(iv) CDRL3 comprises an amino acid sequence: QQ-X1-X2-X3- (X4) n-X5-X6-T (SEQ ID No.:_), wherein X1-X6 are naturally present amino acids other than cysteine and X1 is position 91 according to the Kabat numbering system.

In one aspect, X1 is position 91 according to the Kabat numbering system and X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from P and L; and X 6 is selected from F, L, I and V. In one aspect, n has a value from 1 to 3. In one aspect of the invention, aCDRL3 comprises at least one selected amino acid sequence of any of SEQ ID NOS: 83-188, 507-523, 789-815, and 879-923 or one of the CDRL3 sequences in Figure 11, 15, 21, or 24. In one aspect, the first hypervariable consensus sequence is RASQDVNTAVA (SEQ ID No: 29). In one aspect, the second conservative consensus sequence is S-A-S-L-Y-S (SEQ ID No: 30).

In another example, a polypeptide comprising an immunoglobulin light chain variable domain is provided, in which:

(i) CDRL1 comprises a first hypervariable consensus sequence or variant thereof containing a substitution in one or more positions as compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprises a second hypervariable consensus sequence or variant thereof containing a substitution in one or more positions, as compared to the corresponding hypervariable consensus sequence, and

(iii) CDRL3 comprises an amino acid sequence: Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: _), wherein X1-X5 are amino acids

naturally present other than cysteine and X1 is position 91 according to Kabat numbering system.

In one aspect, X1 is position 91 according to the Kabat numbering system and X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S. In another aspect of the invention, X1 is position 91 according to the Kabat numbering and amino acids at each of the positions of X1-X5 are selected from S and one of Y, W, R eF. In one aspect of the invention, CDRL3 comprises at least one amino acid sequence selected from any of SEQ ID Nos: 83-188, 507-523, 789-815 and 879-923 or one of the CDRL3 sequences in Figure 11, 15, 21. or 24. In one aspect, the first hypervariable consensus sequence is RASQDVNTAVA (SEQ ID No: 29). In one aspect, the second hypervariable consensus sequence is S-A-S-S-L-Y-S (SEQ ID No: 30).

In a specific embodiment, a polypeptide comprises at least two anti-antibody variable domains comprising: providing (a) an antibody heavy chain variable domain comprising any of the above-mentioned heavy chain polypeptide, and (b) a light chain variable domain of antibody comprising any light chain polypeptide cited above.

In certain embodiments, antibodies are provided which include a polypeptide composed of an immunoglobulin heavy chain variable domain according to any of the above cited heavy chain polypeptides, and a polypeptide composed of an immunoglobulin lightweight variable domain according to any one. of the light chain polypeptides mentioned above.

In certain aspects, the above polypeptides and antibodies further include a C-terminal linked dimerization domain of an antibody heavy chain variable domain. In some of these aspects, the dimerization domain comprises a deleucine zipper domain or a sequence that includes at least one cysteine residue. In some of these aspects, the dimerization domain includes a hinge region of an antibody and a leucine zipper. In other respects, the dimerization domain is a single cysteine.

In one example, there is provided a fusion polypeptide comprising any polypeptide cited above, in which an antibody variable domain comprising the polypeptide cited above is fused by at least a portion of a viral capsid protein. In one aspect, the viral capsid protein is selected from the group consisting of the p11 protein, pVIII main protein, Soe, Hoc, gpD, pv1, and their variant. In one aspect, the fusion polypeptide further includes a dimerization domain between the variable domain and capsideviral proteins. In such an aspect, the variable domain is a heavy string variable domain. In another aspect, the fusion polypeptide further includes a variable domain fused to a marker peptide. In such an aspect, the variable domain is a light chain variable domain. In another aspect, the labeled peptide is selected from the group consisting of gD, c-myc, poly-his, proteinfluorescent and 0-galactosidase.

In one example, one or more of the above described polypeptides additionally have support regions FR1, FR2, FR3, and / or FR4 for a variable domain polypeptide corresponding to the CDRH1, CDRH2, CDRH3, and / or CDRL3 variant, where support regions obtained from a single template polypeptide. In such examples, each of the support regions comprises an amino acid sequence corresponding to the support region of the 4D5 antibody amino acid sequences (SEQ ID NO: 6 - 9 and 10-13) or 4D5 antibody variant (SEQID No: 14-17e 18 -21).

In one example, a library is provided which comprises a variety of one or more polypeptides described above, wherein the library has at least 1 x 10 4 antibody variable domain sequences.

In another embodiment, a method for generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides including:

(i) CDRH1 comprises an amino acid sequence: G-F-X1-I-X2-X3-X4-X5-I-H (SEQ ID No.:_), characterized in that G is position 26

and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: _) wherein X1 is position 50 according to the system Kabat numbering; wherein amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S X4 is selected from Y and S; X5 is selected from Y and S; and X6 is selected from YeS.e;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( Wherein X1 is position 95 according to the Kabat numbering system, wherein the amino acids at each of the X1-X6 positions are selected from an amino acid pool at a molar ratio of 50. % Y, 25% S and 25% G, and in which the amino acids at each of the positions of X7-X17 are selected from an amino acid pool at a molar ratio of 50% Y, 25% S and 25%. % of G, or not present, where X18 is selected from G and A and X19 is selected from F, L, I, and M.

In one aspect of the invention, the method comprises:

(b) produce a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprising an amino acid sequence: Q-Q-X1-X2-X3-X4-X5-T, wherein X1 is position 91 according to the Kabat numbering system; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; where X5 is selected from Y and S.

In one aspect of the invention, the variety of polypeptides is encoded by a variety of polynucleotides.

In another example, the method for producing a composition comprises providing a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprising an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No.:_), characterized in that G is 28 according to the numbering system from Kabat; wherein amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S X4 is selected from Y and S; and X5 is selected from Y and S, and;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 1), characterized by the fact that X1 is position 50 according to with the Kabat numbering system, wherein amino acid X1 is selected from Y and S; X2 is selected from Y and S X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Ye S; and X6 is selected from YeS, and;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID No.:_), characterized in that X1 is position 95 according to the Kabat numbering system, in which the amino acids at each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio of 25. % Y, 50% S and 25% R, and wherein the amino acids at each of the X7-X17 positions are selected from a pool of amino acids at a molar ratio of 25% Y, 50% S and 25%. % of R, where X18 is selected from G and A and X19 is selected from I, M, L and F.

In one aspect of the invention, the method comprises:

(b) produce a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprises the amino acid sequence QQ-X1-X2-X3-X4-P-X5-T (SEQ ID No.:_), wherein X1 is position 91 according to Kabat numbering and is selected from S and Y; where X2 is selected from If Y; wherein X3 is selected from Y and S; where X4 is selected from Y and S, where X5 is selected from Y and S.

In one aspect of the invention, the variety of polypeptides is encoded by a variety of polynucleotides.

In another example, the method for producing a composition comprises providing a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprising an amino acid sequence:

G-F-X1-I-X2-X3-X4-X5-I-H (SEQ ID No.:_), characterized in that G

is position 26 and X1 is position 28 according to the Kabat numbering system; wherein amino acid X1 is selected from Y and S; X2 is selected from Ye S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S, and;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 1), characterized by the fact that X1 is position 50 according to with the Kabat numbering system;

wherein amino acid X1 is selected from Y and S; X2 is selected from Y and S X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Ye S; and X6 is selected from Y and S, and;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( Where X1 is position 95 according to the Kabat numbering system, characterized in that the amino acids at each of the positions of X1-X6 are selected from one poo / amino acid in a molar ratio. 38% Y, 25% S, and 25% G and 12% R, characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 38% of Y, 25% S, and 25% G and 12% R, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F.

In one aspect of the invention, the method comprises:

(b) produce a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprises the amino acid sequence QQ-X1-X2-X3-X4-P-X5-T (SEQ ID No.:_), wherein X1 is position 91 according to Kabat numbering and is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; where X4 is selected from Y and S, where X5 is selected from Y and S.

In one aspect of the invention, the variety of polypeptides is encoded by a variety of polynucleotides.

In another example, the method for producing a composition comprises providing a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprising an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No.:_), characterized in that G is position 26 and X1 is position 28 according to with the Kabat numbering system; wherein amino acid X1 is selected from Y and S; X2 is selected from Ye S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from YeS.e;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 1), characterized by the fact that X1 is position 50 according to with the Kabat numbering system, wherein amino acid X1 is selected from Y and S; X2 is selected from Y and S X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Ye S; and X6 is selected from Y and S, and;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( Where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio of 20. % Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1 % K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V and 1% W, or not present; wherein X18 is selected from G eA, and in which X19 is selected from I, M, L and F. In one aspect of the invention, the method comprises:

(b) in the production of a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprises the amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID No.:_), wherein X1 is position 91 according to

Kabat numbering and is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; where X4 is selected from Y and S, where X5 is selected from Y and S.

In one aspect, the first hypervariable consensus sequence comprises a Kabat CDRL1 consensus sequence. In one aspect the first consensus sequence is R-A-S-Q-D-V-N-T-A-V-A (SEQ ID NO: 29). In one aspect, the second hypervariable consensus sequence comprises a Kabat CDRL2 consensus sequence. In one aspect, the second hypervariable consensus sequence is S-A-S-S-L-Y-S (SEQ ID No: 30). In one aspect of the invention, the variety of polypeptides is encoded by a variety of polynucleotides.

In one example, the method for producing a composition comprises providing a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), wherein X1 is position 28 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y;

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to with the Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y X5 is selected from S and Y and X6 is selected from S and Y, and;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-x6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ Where X1 is position 95 according to Kabat numbering system, and wherein X1 is selected from Y, S, G and R; X2 is selected from Y, S, G, R and M; X3 is selected from G, Y, S, R and W; X4 is selected from Y, S, G, R and F; X5 is selected from G, Y, N and A; X6 is selected from F, M, L, A, R, G, H, W, V, Y and S; X 7 is selected from M. L, G, A, R, F and Y; X 8 is selected from M, L, F, I, R, G, P, V, Y and S; X9 is selected from G, Y and R; X10 is selected from M, F, G, Y, R and S; X11 is selected from A, G, Y, R and S; X 12 is selected from I, M, L, F, A, G, R, T, Y and S; X13 is selected from F, M, L, G, A, T, Y and S; X 14 is selected from L, F, M, 1, G, A, Te Y; X15 is selected from M, Y G, L and R or is not present; X16 is selected from Y and G, or not present; X17 is selected from R, M and G, or is not present; X18 is selected from P and A, or not present; and X19 is A, or is not present.

In one aspect of the invention, the CDRH1 comprises at least one amino acid sequence selected from any of IDN SEQs: 189-294. In one aspect of the invention, the CDRH2 comprises at least one amino acid sequence selected from any of IDN SEQs: 295-400. In one aspect of the invention, CDRH3 comprises at least one amino acid sequence selected from any of IDN SEQs: 401-506.

In one aspect of the invention, the method comprises:

(b) in the production of a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprising an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: 25); characterized by the fact that X1 is position 91 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S, Y and F; X3 is selected from Y, S and F; X4 is selected from Y and S, and X5 is selected from S and Y.

In one aspect of the invention, the variety of polypeptides is encoded by a variety of polynucleotides.

In one example, a method is provided for producing a composition comprising a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprising an amino acid sequence:

G-F-X1-I-X2-X3-X4-X5-I-H (SEQ ID No.:_), characterized in that Gé position 26 and X1 is position 28 according to the Kabat numbering system; wherein amino acid X1 is selected from Y and S; X2 is selected from Ye S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S, and (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: _), characterized by the fact that X1 is position 50 according to the Kabat numbering system;

wherein amino acid X1 is selected from Y and S; X 2 is selected from Y and S;

X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Ye S; and X6 is selected from Y and S, and;

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 (SEQ ID No.:_), characterized in that X1 is position 95 according to the Kabat numbering system, and in which the amino acids at each of the positions of X1-X17 are selected from A, C, F, G , I, L, N, P, R, T, W or Y, or are not present; X18 is selected from G and A; eX19 is selected from F, L, I, and M.

In one aspect of the invention, the method comprises:

(b) the production of a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprising an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: _); characterized in that X1 is position 91 according to the Kabat numbering system and is selected from Y and S; X2 is selected from Y and S; X3 is selected from Y, S and F; X4 is selected from Y and S, and X5 is selected from Y and S.

In one example, a method is provided for producing a composition comprising a variety of polypeptides, comprising:

(a) producing a variety of polypeptides including:

(i) CDRH1 comprising an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No.:_), characterized in that G is position 26 and X1 is position 28 according to with the Kabat numbering system; and wherein the amino acids at each of the positions of X1-X5 are selected from S and one of Y, W, R or F; and

(ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 1), characterized by the fact that X1 is position 50 of according to the Kabat numbering system; and wherein the amino acids at each of the positions of X1-X6 are selected from S and one of Y, W, R or F; and

(iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 (SEQ ID No.:_), characterized in that X1 is position 95 according to the Kabat numbering system, and the amino acids in each of the X1-X17 depositions are selected from S and one of Y, W, R or F is not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M.

In one aspect of the invention, the method comprises:

(b) the production of a variety of polypeptides including:

(i) CDRL1 comprising a first hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence;

(ii) CDRL2 comprising a second hypervariable consensus sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and;

(iii) CDRL3 comprising an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: _); characterized by the fact that X1 is apposition 91 according to the Kabat numbering system and the amino acids at each of the positions of X1-X5 are selected from S and one of Y, W, R or F.

In one example, a method for producing one or more antibody CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences described above is provided as follows:

(a) constructing an expression vector containing a polynucleotide sequence encoding a light chain, a heavy chain, or both light chain and heavy chain variable domains of an anti-source including at least one, two, three, four, five, or all selected CDRs of the group consisting of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3; and

(b) mutating at least one, two, three, four, five or all antibody CDRs to generate one or more of the hypervariable regions described above.

In one example, a method for selecting a polypeptide that binds to a target antigen is provided, comprising:

(a) generating a composition with a variety of one or more polypeptides described above;

(b) selecting one or more polypeptides of the target antigen-binding composition;

(c) isolating one or more antigen binding polypeptides non-binding antigen polypeptide targets; and

(d) identifying one or more target antigen-binding polypeptides with a desired target antigen-binding affinity. In one example, a method is provided for selecting a variable domain that binds to target antigens of an antibody library, comprising:

(a) interacting one or more of the above described libraries with a target antigen;

(b) separating one or more polypeptides that specifically bind to the target antigen from polypeptides that do not specifically bind to the target antigen by recovering one or more polypeptides that specifically bind to the target antigen and incubating one or more polypeptides that specifically bind to the target antigen in a series of solutions. which consists in decreasing the amount of target antigen to a concentration of about 0.1 nM to about 1000 nM, and

(c) Selection of one or more polypeptides that specifically bind to the target antigen and which may bind to a low target antigen concentration or that have an affinity of from about 0.1nM to 200nM.

In one aspect, the target antigen is HER2 or DR5. In one aspect, the target antigen concentration is about 100 to 250 nM. In one aspect, the target antigen concentration is about 25 nM to 100 nM. In some embodiments, one or more libraries, clones, or polypeptides are selected against an antigen panel that includes the target antigen. In some embodiments, clones or polypeptides that specifically bind to the target antigen and do not cross-target with any other panel antigen are selected. The antigen panel may include at least three or above 100 antigens. In some cases, the antigen panel includes from 3 to 100, from 3 to 50, from 3 to 25, from 3 to 10 different antigens.

In one example, a method is provided for selecting a polypeptide that binds to the target antigens of a polypeptide library, comprising:

(a) isolating one or more polypeptides that specifically bind to the target antigen by interacting with a library containing a variety of polypeptides described above with a target antigen immobilized under conditions suitable for binding;

(b) separating one or more specifically binding antigen-binding polypeptides from non-specifically binding antigen, and recovering one or more polypeptides that specifically bind to the target antigen to obtain an enriched subpopulation for one or more specifically binding polypeptides target antigen; and

(c) optionally repeating steps (a) - (b) at least twice, each repeating using the enriched subpopulation for one or more polypeptides that specifically bind to the target antigen obtained from the last round of selection.

In one aspect, the method further comprises:

(d) incubating the subpopulation with a labeled target antigen concentration within a range of about 0.1 nM to 1000 nM to form a mixture under conditions suitable for binding;

(e) interacting the mixture with an immobilized agent that binds labeled target antigen;

(f) detecting one or more polypeptides that specifically bind to the labeled target antigen, and recovering one or more polypeptides that specifically binds to the labeled target antigen from the labeled target antigen; and

(g) optionally repeating steps (d) to (f) for at least two times, each repetition using the enriched subpopulation for one or more polypeptides that specifically bind to the target antigen obtained from the last round of selection, using a lower target antigen concentration marked than in the previous selection cycle.

In one aspect, the method further comprises adding an excess of unlabeled target antigen to the mixture and incubating the mixture for a time sufficient to recover one or more polypeptides that specifically bind to the low affinity target antigen. In some embodiments of any of the methods described herein, one or more libraries, clones or polypeptides are selected against an antigen panel including the target antigen. In some exemplary embodiments, clones or polypeptides that specifically bind to the target antigen and do not cross react with any other panel antigen are selected. The antigen panel may include at least three or above 100 antigens. In some cases, the antigen panel includes 3 to 100, 3 to 50, 3 to 25, 3 to 10 different antigens.

In one example, a method for isolating one or more polypeptides that specifically bind to a high affinity target antigen is provided, comprising:

(a) Interacting a library containing a variety of any polypeptide described above with a target antigen at a concentration of at least 0.1 nM to 1000 nM to isolate one or more polypeptides that specifically bind to the target antigen;

(b) recovering one or more polypeptides that specifically bind to the target antigen from the target antigen to obtain an enriched subpopulation for one or more polypeptides that specifically bind to the target antigen; and

(c) optionally repeating steps (a) and (b) at least twice, each repeating using the subpopulation obtained from the last step and using a decreasing target antigen concentration than used in the previous step to isolate one or more polypeptides that are specifically bind to the target antigen at a lower concentration of target antigen.

In one example, a method is provided for selecting one or more polypeptides that specifically bind to a target antigen from a library containing a variety of polypeptides described above comprising:

(a) Interacting a library with a labeled target antigen concentration at a concentration of about 0.1 nM to 1000 nM under ideal conditions for the formation of one or more complexes between the labeled target antigen and one or more polypeptides that bind to the target antigen. ;

(b) isolating one or more polypeptides that specifically bind the target antigen from the labeled target antigen to obtain an enriched subpopulation for one or more polypeptides that specifically bind to the target antigen; and

(c) optionally repeating steps (a) and (b) at least twice, each repeating using the subpopulation obtained from the last step and using a decreasing target antigen concentration that was used in the previous step.

In one aspect, the assay further comprises adding an excess of unlabeled target antigen to one or more complexes. In one aspect, steps (a) and (b) are repeated twice in which the target antigen concentration in the first selection step is about 100nM to 250 nM, where the target antigen concentration in the second selection step is about from 25 nM to 100 nM, and wherein the alvone antigen concentration second selection step is about 0.1 nM to 25 nM.

In one embodiment, a method is provided for selecting a library containing a variety of any of the above described polypeptide, comprising:

(a) incubating a first library sample with a target antigen under conditions suitable for binding of the polypeptides to the target antigen;

(b) incubating a second library sample in the absence of a target antigen;

(c) interacting each of the first and second sample with an immobilized target antigen under conditions suitable for binding of polypeptide to the immobilized target antigen;

(d) detecting target antigen bound polypeptides for each sample, and

(e) determining the affinity of binding of the polypeptide to the target antigen by calculating the ratio of the amount of bound polypeptide from the first sample to the amount of bound polypeptide from the second sample.

In one aspect, the target antigen is DR5 or HER2. In one aspect, the target antigen concentration is about 100 to 250 nM. In one aspect, the target antigen concentration is about 25 nM to 100 nM. In some embodiments, one or more libraries, clones, or polypeptides are selected against an antigen panel that includes the target antigen. In some embodiments, clones or polypeptides that specifically bind to the target antigen and do not cross-target with any other panel antigen are selected. The antigen panel may include at least three or above 100 antigens. In some cases, the antigen panel includes from 3 to 100, from 3 to 50, from 3 to 25, from 3 to 10 different antigens.

In one example, one or more of the polypeptides described above specifically bind to human DR5. In one aspect, opolipeptide is an antibody that specifically binds to human DR5. In one aspect, the antibody comprises the framework region of the 4D5 antibody. In one aspect, the antibody comprises the framework regions of a variant 4D5 antibody. In such an aspect, the antibody is a monoclonal antibody. In such respect, the antibody is a bispecific antibody. In such an aspect, the antibody is a synthetic antibody.

In one example, the anti-DR5 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQID NO : 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; wherein X3 is selected from Ye S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is apposition 50 according to Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Ye S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X.14-X15-X16-DY (SEQ ID NoED 31), wherein X1 is position 95 according to the Kabat numbering system, and wherein X1 is selected from R, Y, M; X 2 is selected from Y and R; X3 is selected from Y, S, R, P and G; X4 is selected from Y and S; X5 is selected from Y, S, R and H; X6 is selected from R, Y and S; X7 is selected from G, Y and S; X8 is selected from R, Y and S; X9 is selected from G, Y and S; X10 is selected from R, Ye S; X11 is selected from G, Y and S; X 12 is selected from S, Y, R, G and A; X13 is selected from G and Y; X 14 is selected from L, M, R, G and A; and X15 is selected from G, F and L or is not present, and X16 is F or not present.

In another example, the anti-DR5 antibody comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQID No: 23), wherein X1 is position 50 according to the system Kabat numbering; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Ye S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID No ED: 24), where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each position of X1-X6 are selected from an amino acid pool at a molar ratio of 50% Y, 25% S, and 25% G, characterized in that the amino acids at each of the X7-X17 positions are selected from an amino acid pool at a 50% Y, 25% molar ratio. S and 25% G, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F.

In another example, the anti-DR5 antibody comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQID No: 23), wherein X1 is position 50 according to the system Kabat numbering; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Ye S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 26), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids in each of the positions of X1-X6 are selected from a pool of amino acids in a molar ratio of 25. % Y, 50% S, and 25% R; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 25% Y, 50% S and 25% R, or not present; wherein X18 is selected from G and A; and wherein, X19 is selected from I, M, Le F.

In another example, the anti-DR5 antibody comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQID No: 23), wherein X1 is position 50 according to the system Kabat numbering; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Ye S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 27), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each position of X1-X6 are selected from an amino acid pool at a molar ratio of 38% Y, 25% S, 25% G and 12% R; characterized in that amino acids at each of the X7-X17 positions are selected from a pool of amino acids in a molar ratio of 38% Y, 25% S, and 25% G and 12% R, or not present; wherein X18 is selected from G and A, wherein X19 is selected from I, M, L and F.

In another example, the anti-DR5 antibody comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQID No: 23), wherein X1 is position 50 according to the system Kabat numbering; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Ye S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( In which: X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each position of X1-X6 are selected from an amino acid pool at a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V and 1% W, characterized by the fact that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D , 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q , 1% T, 1% V and 1% W, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F.

In some embodiments, the anti-DR5 antibody has a CDRH1 which comprises at least one selected amino acid sequence of any of SEQ ID Nos: 524-540 as shown in Figure 15. The anti-DR5 antibody may also have a CDRH2 which comprises at least least one amino acid sequence selected from any of SEQ ID Nos: .541-557 as shown in Figure 15. The anti-DR5 antibody may also have a CDRH3 comprising at least one amino acid sequence selected from any of SEQ ID Nos: .558- In one aspect, an antibody that specifically binds to human DR5 comprises CDRH1, CDRH2, CDRH3 and CDRL3 consequences corresponding to the CDRH1, CDRH2, CDRH3 and CDRL3 sequences set forth in Figure 15 for any of the 1-17 Fabs.

In some embodiments, the anti-DR5 antibody had a CDRH1 in which the position of amino acid X1 is position 95 and is selected from R and Y; X3 is position 97 and is S; X8 is amino acid position 100b and is S; X9 is the position of amino acid 100c and is Y; X10 e is apposition of amino acid 100d and is Y or R. In one example, CRDH3 has the amino acid sequence X1-R-S-Y-R-Y-G-Y-X10-G-S-Y-X14-F-D-Y (SEQ ID NO: 575). In a specific embodiment, an anti-DR5 antibody has a heavy chain variable domain comprising: i) a CDRH1 containing a GFYISSSSIH amino acid sequence (SEQ ID No: 576); ii) a CDRH2 containing a SISPSSGSTYYADSVKG amino acid sequence (SEQ NO ID: 577); and iii) a CDRH3 containing an amino acid sequence YRSYRYGSYYGSYGFDY (SEQ NOID: 578). In another specific example, an anti-DR5 antibody has a heavy chain variable domain comprising: i) a CDRH1 containing a GFYISSSSIH amino acid sequence (SEQ ID No: 579); ii) a CDRH2 containing an amino acid sequence SISPSSGYTSYADSVKG (SEQ NO ID: 580); and iii) a CDRH3 containing an amino acid sequenceRRSYRYGSYRGSYAFDY (SEQ NO ID: 581).

In some embodiments, the anti-DR5 antibody had a CDRH1 comprising an amino acid sequence GFXIIX2SSSIH (SEQ ID NO: 598) wherein X1 and X2 are Y or S. In another embodiment, the anti-DR5 antibody had a CDRH2 comprising an amino acid sequence X1ISPX3X4GYTX6YADSKVG (SEQ ID NO: 599) and in which, X1, X2, X4 and X6 are Y or S. In another embodiment, the anti-DR5 antibody has a CDRH3 comprising an amino acid sequenceYRX3YRYGX8YXXYYXXYYXXYXY 596), wherein X3 is selected from Y, S, R, P and G; X8 is selected from R, Y and S X9 is selected from G, Y and S; X10 is selected from S, Y and R X14 is selected from G and A and X15 is selected from L and F.

In some embodiments, the anti-DR5 antibody binds human DR5 with an IC50 of 1 to 20 nM. In another example, the anti-DR5 antibody binds to murine DR5.

Anti-DR5 antibodies may optionally have a light chain variable domain, in which: (i) CDRL3 comprises an amino acid sequence Q-Q-X1-X2-X3-X4-P-X5-T (SEQ ID NO: 25); wherein X1 is apposition 91 and is selected from Y, H and S; X 2 is selected from Y and S; X3 is selected from Y, S and T; X4 is selected from Y, S and T, and X5 is selected from S, P and Y. Anti-DR5 antibodies may optionally have a light chain variable domain, in which CDRL3 comprises a QQXIX2X3SPST amino acid sequence (SEQ ID NO: 597), wherein X1, X2 and X3 are Y or S. The light chain variable domain may additionally have a CDRL3 which comprises an amino acid sequence of a group consisting of SEQ ID Nos: 507 to 523, as shown in Figure 15. The antibody may additionally comprise a CDRL1 comprising a RASQDVNTAVA (SEQ) amino acid sequence. ID No: 29). The antibody may additionally consist of a CDRL2 comprising an amino acid sequence SASSLYS (SEQ ID No: 30).

In one aspect, DR5-specific antibodies are selected for agonist or antagonist activity. Such antibodies may be selected in a DR5 receptor signaling assay, such as an apoptosis assay as described herein. Antibody antagonists increase apoptosis compared to control and antagonist antibody decreases apoptosis.

In one example, an isolated polynucleotide encoding one of the antibodies described above that specifically binds to human DR5 is provided. In one example, a vector containing an isolated polynucleotide encoding one of the antibodies described above that specifically binds human DR5 is provided. By way of example, a host cell transformed with a vector containing an isolated polynucleotide encoding one of the antibodies described above that specifically binds human DR5 is provided. By way of example, an antibody production process is provided, comprising from a host cell culture transformed with a vector containing an isolated polynucleotide encoding one of the above described antibodies that specifically binds human DR5 such that the polynucleotide is expressed. In one aspect, the process further comprises recovering the antibody from host cell culture. In one aspect, the process further comprises recovering the antibody from the cultured host cell medium.

In one example, there is provided a method of using one or more antibodies described above that specifically bind human DR5 to treat an abnormal angiogenesis-associated disease in a mammal in need of such treatment, the step comprising administering one or more antibodies to the mammal. . In some embodiments, apoptosis inhibiting DR5 antibodies may be useful under conditions in which inhibition of cell death is desired (e.g., macular degeneration). In one aspect, the disorder is cancer. In some embodiments, the anti-DR5 antibody increases apoptosis. In one of these aspects, cancer is selected from breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. In another aspect, the treatment further includes the step of administering a second therapeutic agent simultaneously or sequentially with the antibody. In such an aspect, the second therapeutic agent is selected from an anti-angiogenic agent, anti-neoplastic agent, chemotherapeutic agent and cytotoxic agent.

In one example, a method is provided for treating a mammal suffering or at risk of developing an inflammatory or immune disorder, comprising the step of treating the mammal with one or more Fabs of one or more antibodies described above that specifically bind human DR5. . In one aspect, the disorder is immune inflammatory or rheumatoid arthritis. In some embodiments, the anti-DR5 antibody increases apoptosis.

The methods described herein also provide for an isolated HER2 antibody. In an example embodiment the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is apposition 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-DY (SEQ ID NO: 582), wherein X1 is the position of amino acid 95 according to the numbering system from Kabat and is selected from S and Y; X2 is selected from S and Y and R; X3 is selected from S, G, Y and H; X4 is selected from S, G, Y and R; X5 is selected from G and A; X6 is selected from F, M, L and A; and X7 is selected from F, M and L or is missing.

In another embodiment, the anti-HER2 antibody contains an immunoglobulin heavy chain variable domain comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQID No: 22) where X1 is position 28 according to the Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50, according to Kabat's numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from If Y; X5 is selected from S and Y, and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X7-X8-DY (SEQ ID NO: 583), where X1 is the position amino acid 95 according to the Kabat numbering system and is selected from Y, S and G; X 2 is selected from Y, S, G, R and A; X3 is selected from G, Y, S and R; X4 is selected from G, Y and F; X5 is selected from Y, S, N and G; X6 is selected from Y, R, H and W; and X7 is selected from G and A; and X8 is selected from F, M, L and I.

In another embodiment, the anti-HER2 antibody contains an immunoglobulin heavy chain variable domain comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQID No: 22) where X1 is position 28 according to the Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50, according to Kabat's numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y, and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16- X17-X18-X19-DY (SEQ ID NO: 584), wherein X1 is amino acid position 95 and is selected from Y, S, R and G; X 2 is selected from Y, S, R and G; X3 is selected from S, Y, G and W; X4 is selected from S, Y, G and Q; X5 is selected from G, Y and S; X6 is selected from G, Y S, R and V; X7 is selected from S, Y, G and R; X8 is selected from Y, S, G, R, P and V; X9 is selected from G, A, Y, S and R; X10 is selected from M, F, G, Y, S and R; X11 is selected from A, Y, S, G and R; X12 is selected from I, M, F, L, A, G, S, Y, R and T; X13 is selected from F, M, L, G, A, Y, T and S or is not present; X 14 is selected from L, M, F, I, G, Y, A and T, and X15 is selected from M, L, Y, G and R or is not present; X16 is selected from Y and G or is not present; X17 is selected from R, M and G is not present; X18 is selected from P and A or is not present; eX18 is L or not present.

In yet another example, the anti-HER2 antibody from an immunoglobulin heavy chain variable domain comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID No: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQID No: 23), wherein X1 is position 50 according to the system Kabat numbering; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; wherein X5 is selected from Ye S, and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1 -X2-X3-X4-X5-x6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 24), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids in each of the positions of X1-X6 are selected from a pool of amino acids in a molar ratio of 50. % Y, 25% S, and 25% G; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 50% Y, 25% S and 25% G, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F.

In yet another example, the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from If Y; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to the system Kabat numbering; where X1 is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S, and where X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 26), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from a pool of amino acids in a molar ratio of 25. % Y, 50% S, and 25% R; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of 25% Y, 50% S and 25% R, or not present; wherein X18 is selected from G and A; and wherein, X19 is selected from I, M, L and F.

In yet another example, the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from If Y; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to the system Kabat numbering; where X1 is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S, and where X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( SEQ ID NO: 27), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids in each of the positions of X1-X6 are selected from a pool of amino acids in a molar ratio of 38. % Y, 25% S, and 25% G and 12% R; characterized in that the amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio 38% Y, 25% S, and 25% G and 12% R, or not wherein X18 is selected from G and A, and wherein X19 is selected from I, M, L and F.

In yet another example, the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from If Y; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to the system Kabat numbering; where X1 is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S, and where X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY ( In which: X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each position of X1-X6 are selected from an amino acid pool at a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1 % deK, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; characterized in that the amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio 20% Y, 26% S, 26% G, 13% R, 1% A , 1% D, 1% E, 1% F, 1% H, 1% 1.1% K, 1% L, 1% M, 1% N, 1% P 1% Q, 1% T, 1% V and 1% W, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F.

In yet another example, the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from If Y; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to the system Kabat numbering; where X1 is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S, and where X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 (SEQ ID No.:_), characterized in that X1 is apposition 95 according to the Kabat numbering system, and in which amino acids at each of the positions of X1-X19 are selected from A, C, F, G, 1, L, N, P, R, T, W or Y, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M is not present.

In yet another example, the anti-HER2 antibody comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from If Y; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), wherein X1 is position 50 according to the system Kabat numbering; where X1 is selected from Y and S; wherein X2 is selected from Ye S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S, and where X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-

X16-X17-X18-X19 (SEQ ID No.:_), characterized in that X1 is apposition 95 according to the Kabat numbering system, and amino acids at each of the positions of X1-X19 are selected from If one of Y, W, R, or F is not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M.

In some embodiments of the invention, the anti-HER2 antibody may consist of a CDRH1 comprising at least one amino acid sequence selected from a group consisting of one of SEQ ID Nos: 189-294 as shown in Figure 11. The anti-HER2 antibody HER2 may also consist of a CDRH2 comprising at least one amino acid sequence selected from a group consisting of one of SEQ ID Nos: 295-400, as shown in Figure 11. Anti-HER2 antibody may also consist of a CDRH3 comprising at least of an amino acid sequence of a group consisting of one of SEQ IDNos: 401-506 as shown in Figure 11. In some embodiments of the invention, the anti-HER2 antibody may consist of a CDRH1 comprising at least one selected amino acid sequence of a group consisting of one of SEQ ID Nos: 816-842 as shown in Figure 21. The anti-HER2 antibody may also consist of a CDRH2 comprising at least one amino acid sequence selected from a group consisting of one of SEQ ID Nos: 843-869, as shown in Figure 21. The anti-HER2 antibody may also consist of a CDRH3 comprising at least one amino acid sequence from a group consisting of one of SEQs ID Nos: 870-896 as shown in Figure 21. In some embodiments of the invention, the anti-HER2 antibody may consist of a CDRH1 comprising at least one amino acid sequence selected from a group consisting of one of SEQ IDNs: 924-950 as shown in Figure 24A. The anti-HER2 antibody may also contain a CDRH2 comprising at least one amino acid sequence selected from a group consisting of one of SEQ ID Nos: 951-977, as shown in Figure 24A. The anti-HER2 antibody may also consist of a CDRH3 comprising at least one amino acid sequence from a group consisting of one of SEQ IDNs: 978-1004 as shown in Figure 24A.

In one aspect, an antibody that specifically binds to human HER2 comprises the CDRH1, CDRH2, CDRH3 and CDRL3 sequences corresponding to the CDRH1, CDRH2, CDRH3 and CDRL3 sequences set forth in Figure 11 for any of the 1-106 Fabs. In one aspect, an antibody that specifically binds to human HER2 comprises the CDRH1, CDRH2, CDRH3 and CDRL3 sequences corresponding to the CDRH1, CDRH2, CDRH3 and CDRL3 sequences set forth in Figure 21A for any of the B1-B28 clones. In another aspect, an antibody that specifically binds to human HER2 comprises the CDRH1, CDRH2, CDRH3 and CDRL3 sequences corresponding to the CDRH1, CDRH2, CDRH3 and CDRL3 sequences set forth in Figure 24A for any of the G29 to G61 clones.

In some embodiments, an anti-HER2 antibody has a CDRH1 comprising an amino acid sequence GFSIX2X3SYIH (SEQ ID NO: 588) wherein X2 and X3 are Y or S. An anti-HER2 antibody has a CDRH2 comprising an amino acid sequenceSIYPX3SGYTSYADSKVG Wherein X3 is Y or S. An anti-HER2 antibody may additionally have a decade-long CDRL1 variable domain comprising an amino acid sequence QQSYYX4PST (SEQ ID NO: 587), wherein X4 is Y or S.

In some embodiments, an anti-HER2 antibody has an amino acid sequence GFX1ISYSSIH (SEQ ID No: 590) wherein X1 is Y or S. An anti-HER2 antibody additionally has a CDRH2 comprising an amino acid sequence SIYPX3YGX5TX6YADSKVG 591) and in which X3, X5 and X6 are Y or S.

In some embodiments, an anti-HER2 antibody has a CDRH1 comprising an amino acid sequence GFXIISSSSIH (SEQ ID No: 593) wherein X1 is Y or S. An anti-HER2 antibody may additionally have a CDRH2 which has an amino acid sequence X1DC2PSSGYTX6YADSKVG Wherein X1, X2, and X6 are Y or S. An anti-HER2 antibody may additionally have a CDRH3 which has an amino acid sequenceXEX2X3X4YYSYYX10GX12X13X14DY (SEQ ID NO: 592) and wherein, X1 is selected from Y, S and R, X 2 is selected from Y and S; X3 is selected from G, Ye S; X4 is selected from G, Y and S; X4 is selected from Y, S, R and G; X10 is selected from Y, S and G, X12 is selected from Y, S and G; X13 is selected from G and A and X14 is selected from I, F, M and L.

In some embodiments, the anti-DR5 antibody may optionally have a light chain variable domain in which: CDRL3 comprises an amino acid sequence QQ-X1-X2-X3-X4-P-X5-T (IDSEQ NO: 25) ; where X1 is position 91 and is selected from S and Y; X 2 is selected from S, Y and L; X3 is selected from Y, S and F; X4 is selected from Y and S, and X5 is selected from S and Y. The light chain variable domain may additionally have a CDRL3 comprising an amino acid sequence of a group consisting of SEQs ID Nos: 83 to 188, as shown in Figure 11, SEQs ID Nos: 789 to 815 as shown in Figure 21A and SEQs ID Nos: 897 to 923 as shown in Figure 24A. The antibody may additionally consist of a CDRL1 comprising an amino acid sequence RASQDVNTAVA (SEQ ID NO: 29). The antibody may further comprise a CDRL2 comprising a SASSLYS amino acid sequence (SEQ ID No: 30).

In one aspect, the polypeptide is a specific antibody that selects HER2. In such an aspect, the antibody comprises the 4D5 antibody structural region. In such an aspect, the antibody comprises the framework region of a 4D5 antibody variant. In such an aspect, the antibody is an anticomponent. In such an aspect, the antibody is a bispecific antibody.

In one example, an isolated polynucleotide encoding one of the antibodies described above that specifically binds to human HER2 is provided. In one embodiment, a vector containing an isolated polynucleotide encoding one of the antibodies described above that specifically binds to human HER2 is provided. For example, a vector transformed host cell containing an isolated polynucleotide encoding one of the above described antibodies that specifically binds to human HER2 is provided. For example, an antibody production process is provided, comprising a vector-transformed host cell culture containing an isolated polynucleotide encoding one of the above described antibodies that specifically binds human HER2 such that the polynucleotide is expressed. In one aspect, the process further comprises antibody recovery from host cell culture. In one aspect, the process further comprises recovering the anti-host cell culture medium.

In one example, there is provided a method of using one or more antibodies described above that specifically bind human HER2 to treat a HER2-associated disorder, comprising the step of administering one or more antibodies to the mammal. In another aspect, treatment is the step of administering a second therapeutic agent simultaneously or sequentially with the antibody. In this regard, the second therapeutic agent is selected from an anti-angiogenic agent, antineoplastic agent, chemotherapeutic agent and cytotoxic agent.

In one embodiment, a method of treating a mammal from suffering or at risk of developing a HER2-related disorder includes the step of treating the mammal with one or more Fabs of one or more antibodies described above that specifically bind HER2. In one example, one or more of the polypeptides described above specifically binds HER2.

In one aspect, a polypeptide of the invention includes at least one or both of the heavy and light chain variable domain, wherein the variable anti-antibody comprises one, two, or three variant CDRs as described herein (e.g., as described above).

In one embodiment, a polypeptide of the invention (specifically one comprising an antibody variable domain) additionally has an antibody structural region sequence, for example, FR1, FR2, FR3, and / or FR4 for a variable domain polypeptide corresponding to CDR variant, the FR sequences obtained from a single model antibody. In such examples, FR sequences are obtained from human antibodies. In such examples, the FR sequences are obtained from the human consensus sequence (eg, subgroup III consensus sequence). In one example, the structural region sequences comprise a modified consensus sequence as described herein (for example, comprising modifications at position 49, 71, 93, and / or 94 heavy chain, and / or position 66, of the light chain) . In one example, the framework regions have the framework region sequences of the 4D5-8 antibody (wild type humanized light and heavy chain H 10 (shown in Figure 16 (SEQ IDNos: 6-9 and 10-13, respectively)). In one example, the structural region has sequences of the structural region of a variant version of the light chain and heavy chain humanized antibody 4D5-8, in which the light chain is modified at position 66 and the heavy chain is modified at positions 71, 73 and 78 (as shown in Figure 17 (SEQ ID Nos: 14-17 and 18-21)).

In some embodiments, a polypeptide of the invention comprises a light chain and heavy chain variable domain antibody, wherein the light chain variable domain includes at least 1, 2, or 3 variants selected from the group of CDRs consisting of CDRs. L1, L2 and L3, and the heavy chain variable domain includes at least 1, 2 or 3 variants selected from the group of CDRs consisting of CDRs H1, H2 and H3.

In some examples, a polypeptide of the invention is an ScFv. In some embodiments, it is a Fab fragment. In some embodiments, it is an F (ab) 2 or F (ab ') 2. Thus, in some embodiments, a polypeptide of the invention further includes a dimerization domain. In some embodiments, the dimerization domain is located between an antibody heavy chain or light chain variable domain to at least a portion of a viral capsid protein. The dimerization domain may include a dimerization sequence and / or a sequence that includes one or more cysteine residues. The dimerization domain may be linked, directly or indirectly, to the C-terminal end of a heavy chain or domain constant domain. The structure of the dimerization domain may be varied, depending on whether the antibody variable domain is produced as a component of the fusion protein with the capsideviral protein component (for example, without a stop codon after the dimerization domain) or if The variable domain of the antibody is produced predominantly without the viral capsid protein component (for example, with a codon encountered after the dimerization domain). When the antibody variable domain is produced predominantly as a component protein fusion protein of the viral capsid, one or more disulfide bonds and / or single dimerization sequence provides a bivalent display. For the predominantly produced antibody variable domains not being fused to a viral capsid protein component (e.g., with an amber stop codon), the dimerization domain may include both a cysteine residue and a dimerization sequence. In some embodiments, F (ab) 2 heavy chains dimerize in the dimerization domain not including the hinge region. The dimerization domain may include a deleucine zipper sequence (for example, a GCN4 sequence such asGRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG (SEQ ID No: 3)).

In some embodiments, a polypeptide of the invention further includes a light chain constant chain domain fused to a light chain variable domain, which in some embodiments includes at least one, two or three variant CDRs. In some embodiments of the polypeptides of the invention, the polypeptide comprises a heavy chain constant domain fused to a heavy chain variable domain, which in some embodiments includes at least one or two other variant CDRs.

In some cases, mutation of a structural region residue may be preferable so that it is variant with respect to the reference polypeptide or source antibody. For example, the residue of heavy chain structural region 71 may be R, V or A. In another example, the residue of heavy chain structural region 93 may be S or A. In yet another example, the residue of structural region 94 is R, K or T or is coded by MRT. In another example, the residual of the heavy chain framework 49 may be A or G. The residuals of the heavy chain framework may also be mutated. For example, the amino acid at position 66 could be R or G.

As described herein, a variant CDR refers to a CDR with a different sequence compared to the corresponding CDR sequence of a single reference polypeptide / source antibody.

Accordingly, CDRs of a single polypeptide of the invention may, in certain embodiments, correspond to the CDR set of a single reference polypeptide or source antibody. Invention polypeptides may include any variant or combination of CDRs. For example, a polypeptide of the invention may include a CDRH1 variant and a CDRH2 variant. A polypeptide of the invention may include a CDRH1 variant, CDRH2 variant and a CDRH3 variant. In another example, a polypeptide of the invention may include a CDRH1 variant, CDRH2 variant, CDRH3 variant and the CDRL3 variant. In another example, a polypeptide of the invention comprises a CDRL1 variant, CDRL2 variant and the CDRL3 variant. Any polypeptide of the invention may contain more than one variant of CDRL3. Any polypeptide of the invention may consist of more than one variant of CDRH3. In one embodiment, a polypeptide of the invention comprises one or more CDR variant sequences as illustrated in Figures 7, 19, and 22. In one example, a polypeptide of the invention comprises a or more CDR variant sequences as illustrated in Figure 11A. In one embodiment, a polypeptide of the invention comprises one or more CDR variant sequences as illustrated in Figure 15. In another embodiment, a polypeptide of the invention comprises one or more variant sequences. CDRs as shown in Figures 21A, 21B. In another embodiment, a polypeptide of the invention comprises one or more CDR variant sequences as illustrated in Figure 24A.

Polypeptides of the invention may be complexed with one another. For example, the invention provides a polypeptide complex comprised of two polypeptides, where each polypeptide is a polypeptide of the invention, and wherein one of the mentioned polypeptides includes at least one, two or all variants of CDRs H1, H2, H3, and the other polypeptide comprises one. light chain CDR variant (e.g. CDR L3). A polypeptide complex may consist of a first and a second polypeptide (wherein the first and second are polypeptides of the invention), wherein the first polypeptide includes at least one, two or three light chain CDR variants, and the second polypeptide includes at least one. one, two or three different heavy chain CDRs. The invention also provides polypeptide complexes comprising the same variant CDR sequences. Complex formation can be mediated by any suitable technique, including dimerization / multimerization into a dimerization / multimerization domain as described herein or by covalent interactions (such as via a disulfide bridge) (which in some contexts is part of the dimerization domain, for example, a dimerization domain may contain a leucine zipper sequence and a cysteine).

In another aspect, the invention provides compositions containing polypeptides and / or polynucleotides of the invention. For example, the invention provides a composition comprising a variety of any of the polypeptides of the invention described herein. Said variety may comprise polypeptides encoded by a variety of cholinucleotides generated using a set of oligonucleotides comprising weakening in the coding sequence of a variant amino acid, wherein said weakening of the multiple codon sequences of the restricted codon set encoding the variant amino acid. A composition comprising a polynucleotide or polypeptide or library of the invention may be in the form of a manufactured kit or article (optionally packaged with instructions, buffers, etc.).

In one aspect, the invention provides polynucleotides that encode a polypeptide of the invention as described herein. In another aspect, the invention provides a vector comprising a sequence that encodes a polypeptide of the invention. The vector may be, for example, a replicable expression vector (for example, a replicable expression vector may be an M13, fl, fd, Pf3 phage or derivative thereof, or a lambdoid phage such as lambda, 21, phi80, phi81, 82, 424, 434, etc. or a derivative thereof). The ovector may consist of a promoter region linked to the opolipeptide coding sequence of the invention. The promoter may be suitable for any expression of the polypeptide, for example, the lac Z promoter system, phosphateal alkaline pho A (AP) promoter, bacteriophage 1pl promoter (temperature-sensitive promoter), tac promoter, tryptophan promoter, T7 bacteriophage promoter. Thus, the invention also provides a vector containing a promoter selected from the group consisting of promoters of the systems described above. The polypeptides of the invention may be displayed in any suitable form according to the need and will of the practitioner.

For example, a polypeptide of the invention may be displayed on a superfiviral, for example a phagemid or phagemid viral particle. Thus, the invention provides viral particles comprising a polypeptide of the invention and / or polynucleotides encoding a polypeptide of the invention.

In one aspect, the invention provides a population comprising a variety of polypeptide or polynucleotides of the invention, wherein each type of polypeptide or polynucleotide is a polypeptide or polynucleotide of the invention as described herein.

In some embodiments, the polypeptides and / or polynucleotides are provided as a library, for example, a library comprising a variety of at least about 1x104, 1x105, 1x106.1x107, 1x108 distinct polypeptides and / or polynucleotides or sequences. In another aspect, the invention also provides a library comprising a variety of viruses or viral particles of the invention, wherein each virus or viral particle exhibits a polypeptide of the invention. The inventive library may include a virus or viral particle to display any number of distinct polypeptides (sequences), for example at least about 1x104, 1x105, 1x106, 1x107, 1x108 distinct polypeptides.

In another aspect, the invention provides host cells having a polynucleotide or vector comprising a sequence encoding a polypeptide of the invention.

In another aspect, the invention provides methods for high affinity deletion selection for specific target antigens. In some of these examples, the specific target antigen includes, but is not limited to, HER2 or DR5.

The methods of the invention provide polypeptide populations (e.g., polypeptide libraries (e.g., antibody variable domain)) with one or more diverse CDR regions. These librarians are classified (selected) and / or screened to identify high affinity ligands for the target antigen. In one aspect, the library linker polypeptide is selected to bind to the target antigen, and to have affinity. Opolipeptide ligand selected using one or more of these deletion strategies can then be screened for affinity and / or specificity (it only binds to the target antigen and does not bind to the other antigen).

In one aspect, a method of the invention comprises generating a variety of polypeptides with one or more diverse CDR regions by classifying the variety of polypeptides into linkers to a target antigen by exposing the variety of polypeptides to a target antigen under conditions suitable for binding; separating antigen binders from those that do not seal; isolating the ligands, and identifying the ligands with high affinity ligation (or any ligand that has a desired binding affinity). The affinity of the ligands that bind to the target antigen can be determined using a variety of techniques known in the art, for example ELISA, as described herein. Optionally, the polypeptides may be fused to a labeled polypeptide, such as gD, polyHis or FLAG, which may be used to screen ligands in combination with screening for the target antigen.

Another embodiment provides a method of isolating or selecting an antibody variable domain that binds to a target antigen from an antibody variable domain library, wherein said method comprises: a) exposing a population comprising a variety of immobilized polypeptides of the invention with a target antigen under conditions suitable for binding to occur and isolating the linker polypeptide on the target antigen; b) separating "linker polypeptides" from non-linkers, and eluting ligands from the target antigen; c) optionally repeating steps a and b at least once (in some embodiments at least twice).

In some embodiments, a method may further comprise: d) incubating the subpopulation with a labeled target antigen concentration within a range of 0.1 nM to 1000 nM to form a mixture under conditions suitable for binding; e) interacting the mixture with an immobilized agent that binds on the labeled target antigen; f) detecting one or more polypeptides that specifically bind to the labeled target antigen, and recovering one or more polypeptides that specifically bind to the labeled target antigen from the labeled target antigen; and g) optionally repeating steps (d) to (f) at least once (in some embodiments for at least twice), using each successively lower concentration of labeled target antigen.

Optionally, the method may comprise adding an excess of unlabeled target antigen to the mixture and incubating for a sufficient time to elute low affinity ligands from the labeled target antigen.

In another aspect the invention provides a method for isolating or selecting high affinity binders (or having a desired binding affinity) for a target antigen. In one example, said method comprises: a) exposing a population comprising a variety of immobilized polypeptides of the invention with a target antigen, wherein the antigen is supplied in a concentration amount within a range of 0.1 nM to 1000 nM to isolate the target antigen-binding polypeptide; b) separating the binding polypeptides from the target antigen; c) optionally repeating steps a and b at least once (in some embodiments, at least twice), each time using successively higher levels of labeled target antigen, d) selecting the ligand polypeptide that selects at lower target antigen concentrations by high affinity (or any desired affinity) by incubating the ligand polypeptide with different dilutions of the target antigen and determining the IC50 of the ligand polypeptide, and; e) identifying a linker polypeptide that has a desired affinity for the target antigen. Affinity can be said, for example, about 0.1 nM to 200 nM, 0.5 nM to 150 nM, 1 nM to 100 nM and / or 25 nM to 75 nM.

Another embodiment provides an assay for isolating or selecting a linker polypeptide comprising: a) exposing a population comprising a variety of invention-labeled polypeptides with a target antigen, wherein the antigen is provided in a concentration amount within a range of 0.1 nM to 1000 nM conditions suitable for binding to form a polypeptide ligand complex and labeled target antigen; b) isolating the complexes and separating the ligand opolipeptide from the labeled target antigen; c) optionally repeat steps a and b for at least one time using a lower target antigen concentration at each step. Optionally, the method may further expose the complex comprising the linker polypeptide and the antigen targeting an excess of unlabeled target antigen. In one realization example, the method steps are repeated twice and the target antigen concentration in the first selection step is in the range 100 nM to 250 nM, and in the second selection step (when performed) it is in the 25 nM range. a100 nM, and the third selection step (when performed) is in the range of 0.1nM to 25 nM.

The invention also includes a method for screening a population comprising a variety of polypeptides of the invention, wherein said method comprises: a) incubating a first sample of the polypeptide population with a target antigen under conditions suitable for binding of the polypeptides to the target antigen b) subject a second sample of the polypeptide population similar to an incubation but, in the absence of the target antigen; (c) exposing each of the first and second samples with the immobilized target antigen under conditions suitable for binding of polypeptides to the immobilized target antigen; d) detecting the amount of target antigen-bound polypeptides immobilized on each sample, e) determining the binding affinity of a target antigen-specific polypeptide by calculating the ratio of the amount of specific polypeptide you selected in the first sample to the amount of specific polypeptide you selected in the second sample.

Libraries generated as described herein may also be screened for binding to a specific target and for lack of binding to a non-target antigen. In one aspect, the invention provides a method for screening a polypeptide, such as an invention antibody variable domain, which binds to a specific target antigen from an antibody variable domain library, wherein said method comprises: a) generating a population comprising a variety of invention polypeptides; b) exposing the polypeptide population to a target antigen under conditions suitable for binding; c) separating the library ligand polypeptide from non-ligand polypeptides; d) identifying a specific antigen-binding ligand polypeptide by determining whether the ligand polypeptide binds to an antigen other than the target antigen; e) isolating a specific antigen-specific polypeptide ligand. In some embodiments, step (e) comprises eluting the classified polypeptides from the target antigen, and amplifying a replicable expression vector encoding said polypeptide ligand. In some embodiments, one or more libraries, clones or polypeptides are screened against a panel of antigens including the target antigen. In some examples, clones or polypeptides that specifically bind to the target antigen and do not substantially cross-react with any of the other antigens on the panel are selected. The antigen panel may include a minimum of three and a maximum of 100 different antigens. In some cases, the antigen panel includes from 3 to 100, from 3 to 50, from 3 to 25, or from 3 to 10 different antigens.

Combinations of one of the screening / selection methods described above can be performed with the screening methods. For example, in one example, the binding polypeptides are first selected by binding to an immobilized target antigen. Binding polypeptides that bind to the immobilized target antigen can then be screened for antigen binding and for lack of binding to a non-target antigen. Binder polypeptides that specifically bind to the target antigen can be amplified as needed. These ligand polypeptides may be selected for greater affinity by exposure to a concentration of labeled target antigens to form a complex, at which the concentration of labeled target antigen is from about 0.1 nM to about 1000 nM, and the complexes are isolated by exposure with an agent that binds to the marker on the target antigen. A linker polypeptide may then be eluted from the labeled target antigen and optionally the selection steps are repeated, and at each step a lower concentration of labeled target antigen is used. Binder polypeptide that can be isolated using this selection method can then be screened for high affinity using, for example, a phase solution ELISA as described, for example, in Examples 2 and 4 or other conventional methods known in the art. . Populations of polypeptides of the invention used in the methods of the invention may be provided in any suitable form of screening steps. For example, polypeptides may be free, in soluble form, attached to a matrix, or present on the surface of a phage-like viral particle or phagemid particle. In some embodiments of the invention methods, the variety of polypeptides is encoded by a variety of replicable vectors provided as a library. In the selection / screening methods described herein, vectors encoding ligand polypeptides may be further amplified to provide sufficient amounts of parapeptide polypeptide in repeats of the selection / screening steps (which, as indicated above, are optional in the methods of the invention).

In one embodiment, the invention provides a deletion method for a polypeptide that binds to a target antigen comprising:

a) generating a composition comprising a variety of polypeptides of the invention as described herein;

b) selecting a linker polypeptide that binds to a target antigen from the composition;

c) isolating the binding polypeptide from non-binding polypeptides;

d) identifying the desired affinity ligands from the isolated ligand polypeptides.

In another example, the invention provides a method for selecting an antigen-binding variable domain that binds to a target antigen from an antibody variable domain library comprising:

(a) exposing the invention antibody variable domain library (as described herein) to a target antigen;

b) separating the binders from non-binders, and eluting the binders from the target antigen and incubating the binders in a solution of decreasing amounts of target antigen at a concentration of about 0.1 nM to 1000 nM;

c) selecting binders that can bind at low target antigen concentrations and have an affinity of from about 0.1nM to 200nM.

In some embodiments, the target antigen concentration is about 100 to 250 nM, or about 25 to 100 nM.

In an example embodiment, the invention provides a deletion method for a polypeptide that binds to a target antigen from a polypeptide library comprising:

(a) isolating the linker polypeptide to a target antigen by exposing a library comprised of a variety of polypeptides of the invention (as described herein) with an immobilized target antigen under conditions suitable for binding;

b) separating the target antigen-binding polypeptides from non-binding polypeptides and eluting the target antigen-binding polypeptides to obtain an enriched subpopulation of ligands, and

c) optionally repeating steps a and b at least once (in some embodiments at least twice), each repetition using subpopulation of ligands obtained from the last deselection step.

In some embodiments, the method of the invention may further comprise:

d) incubating the subpopulation of the linker polypeptide with a labeled target antigen concentration within a range of 0.1 nM to 1000 nM under conditions suitable for ligation to form a mixture;

e) interacting the mixture with an immobilized agent that binds to the labeled target antigen;

f) detecting the binding polypeptide that specifically binds to the labeled target antigen and eluting the labeled target antigen binding polypeptide; and

g) optionally repeating steps (d) to (f) at least once (in some embodiments at least twice), each repetition using the binder subpopulation obtained in the previous selection step and using increasingly lower concentrations target antigen than in the previous step.

In some embodiments, these methods typically comprise adding an excess of unlabeled target antigen to the mixture and incubating for a period of time sufficient to elute low affinity ligands from the labeled target antigen. In another embodiment example, the invention provides a method for isolating high affinity binders for the target antigen comprising:

a) exposing a library comprising a variety of polypeptides of the invention (as described herein) with a target antigen at a concentration of 0.1 nM to 1000 nM to isolate the target antigen binding polypeptide;

b) separating the binding polypeptides from the target antigen to obtain an enriched subpopulation of the binding polypeptide; and

c) optionally repeating steps a and b at least once (in some embodiments at least twice), each time using successively lower concentrations of labeled target antigen relative to the previous step to isolate the lower concentration binding ligand polypeptide to the target antigen.

In one aspect, the invention provides a linker polypeptide selection assay from a library consisting of a variety of polypeptides of the invention (as described herein) comprising:

a) expose a library with a target antigen concentration labeled at a concentration of 0.1 nM to 1000 nM under ideal conditions for binding to occur and form a complex with a ligand polypeptide and the labeled target antigen;

(b) isolating the complexes and separating the ligand polypeptides from the labeled target antigen to obtain deligant enriched subpopulation; and (c) optionally repeating steps (a) and (b) at least once (in some embodiments at least twice), each repetition using the ligand subpopulation obtained from the last deselection step and using a decreasing concentration of target antigen that was used in the previous step.

In some embodiments, the method further comprises adding an excess of unlabeled target antigen to the polypeptide and target antigen complex. In some embodiments, the steps set forth above are repeated at least once (in some embodiments, at least two target antigen concentration in the first selection step is in the range 100 nM to 250 nM, and in the second selection step is in the range 25 nM to 100 nM, and the third selection step is in the range 0.1 nM to 25 nM.

In another aspect, the invention also provides a method for screening a library comprising a variety of invention polypeptides, wherein said method comprises in:

a) incubate a first library sample with a target antigen concentration under conditions suitable for binding of polypeptides to the target antigen

b) incubating a second library sample in the absence of the target antigen;

(c) exposing each of the first and second sample with the immobilized target antigen under conditions suitable for binding of the polypeptides to the immobilized target antigen;

d) detecting binding of the polypeptide to the immobilized target antigen on each sample;

e) determining the binding affinity of a specific polypeptide for the target antigen by calculating the ratio of the amount of specific polypeptide that bound in the first sample to the amount of specific polypeptide that bound in the second sample.

Diagnostic use and therapeutic use of the ligand polypeptides of the invention are contemplated. In a diagnostic application, the invention provides a method for determining the presence of a protein of interest comprising exposing a sample suspected of containing the protein to a linker polypeptide of the invention and determining binding of the polypeptide ligand to the sample. For this use, the invention provides a kit comprising the linker polypeptides and instructions for using the linker polypeptides to detect the protein.

The invention further provides: isolated nucleic acids encoding linker polypeptide; a vector having nucleic acids, optionally an operably linked control sequence recognized by a host cell transformed with the vector; a host cell transformed with the vector, a process of producing the linker polypeptide comprising culturing this host cell so that nucleic acid is optionally expressed by recovering the host cell culture linker polypeptide (e.g., from the culture medium host cell).

The invention also provides a composition comprising a binder polypeptide of the invention and a carrier (e.g., a pharmaceutically acceptable carrier) or diluent. This pharmaceutical composition is sterile and may be lyophilized. Also contemplated is the use of the binder polypeptide of the present invention in the manufacture of a medicament for treating an indication described herein. The composition may additionally comprise a second therapeutic agent such as a chemotherapeutic agent, a cytotoxic agent or an anti-angiogenic agent.

The invention further provides a method of treating a mammal, comprising administering an effective amount of the inventive polypeptide ligand to the mammal. The mammal to be treated in the method may be a non-human mammal, for example a primate or rodent suitable for obtaining preclinical data (e.g., mouse, rat or rabbit). Non-human mammals may be healthy (eg in toxicological studies), or may be suffering from a disease being treated with the linker polypeptide of interest. In one embodiment example, the mammal is suffering from a DR5-related disorder. In another embodiment, the mammal is suffering from a HER2-related disorder.

In one example, the mammal is suffering or at risk of developing abnormal angiogenesis (e.g., pathological angiogenesis). In a specific embodiment, the disorder is a cancer selected from the group consisting of colorectal cancer, renal cell carcinoma, ovarian cancer, non-small cell lung cancer (NSCLC), bronchoalveolar carcinoma, and pancreatic cancer. In another embodiment, the disorder is a disease caused by ocular neovascularization, for example, diabetic blindness, retinopathies, especially diabetic retinopathy, age-induced macular degeneration, and rubella. In another example, the mammal to be treated is suffering or at risk of developing an edema (for example, an edema associated with brain tumors, edema associated with a stroke, or an edemacerebral). In another example, the mammal is suffering from or at risk of developing a disorder or disease selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, ascitestrataria, psoriasis, sarcoidosis, arterial arteriosclerosis, sepsis, burns, and pancreatitis. According to another embodiment, the mammal is suffering from or at risk of developing a genitourinary disease selected from the group consisting of polycystic ovary (IP) disease, endometriosis and uterine fibrosis. In one embodiment, the disorder is a disease caused by non-regulation of cell survival (e.g., abnormal amount of cell death) including, but not limited to, cancer, immune system disorders, nervous system disorders, and vascular system disorders. The amount of the inventive binding polypeptide to be administered is a therapeutically effective amount to treat a disorder. In dose selection studies, a variety of doses of the ligand polypeptide may be administered to the mammal. In another example, the therapeutically effective amount of linker polypeptide is administered to a human patient to treat a disorder in the patient.

In one embodiment, the inventive linker polypeptides useful for treating tumors, malignant tumors and other disorders related to abnormal angiogenesis, including inflammatory disorders or immune disorders and / or diabetes or other insulin-related diseases described herein are Fab or scFv antibodies. Therefore, these ligand polypeptides may be used in the manufacture of a medicament to treat an inflammatory disease or immune disease. A mammal that is at or at risk of developing a disorder or disease described herein may be treated by administering a second therapeutic agent simultaneously, sequentially or in combination with a polypeptide (e.g., an antibody) of the present invention. It should be understood that other therapeutic agents besides the second therapeutic agent may be administered to the mammal or used in the manufacture of a medicament for the desired indication.

These polypeptides may be used to understand the role of stromal host cell collaboration in implanted non-host growth, as in mouse models in which human tumors were implanted. These polypeptides may be used in methods for identifying human tumors that may escape therapeutic treatment or by monitoring or observing the growth of a tumor implanted in a rodent or rabbit following treatment with the polypeptide of this invention. The polypeptides of this invention may also be used to study and evaluate the combination of therapies with a polypeptide of this invention and other therapeutic agents. The polypeptides of this invention may be used to study the role of a target molecule of interest in other diseases by administering the polypeptides to an animal suffering from the disease or similar disease and thereby determining if one or more symptoms of the disease are attenuated.

For the sake of clarity, in the description herein, unless otherwise specified or contextually indicated, all amino acid numbers are in accordance with Kabat et al (see further elaboration in "Definitions" below).

Brief Description of the Figures

Figure 1 shows the sequences of a 4D5 heavy chain light chain domain (SEQ ID No: I & 2, respectively).

Figure 2 shows a 3-D model of the humanized 4D5 structure showing the CDR residues that form contiguous shadows. Contiguous shadows are formed by amino acid residues 28, 29, 30, 31 and 32 in CDRL1; amino acid residues 50 and 53 of CDRL2; amino acid residues 91, 92, 93, 94 and 96 of CDRL3; amino acid residues 28, 30, 31, 32.33, no CDRH1; and amino acid residues 50, 52, 53, 54, 56 and 58 in CDRH2.

Figure 3 shows the amino acid frequency (identified by the single letter code) in the human antibody light chain CDR sequences from the Kabat database. The frequency of each amino acid at a given amino acid position is displayed starting with the most frequent amino acid in the left position and continuing to the right for the less frequent amino acids. The number below the amino acid represents the number of naturally occurring sequences in the Kabat database that have that amino acid at that position.

Figure 4 shows the amino acid frequency (identified by the single letter code) in the human antibody heavy chain CDR sequences from the Kabat database. The frequency of each amino acid at a given amino acid position is displayed starting with the most frequent amino acid in the left position and continuing to the right for the less frequent amino acids. The number below the amino acid represents the number of naturally occurring sequences in the Kabat database that have that amino acid at that position. The amino acid positions in the structural region 71, 93 and 94 are also displayed.

Figure 5 schematically illustrates a bicistronic vector allowing the expression of separate transcripts for the display of F (ab) 2. An appropriate promoter directs the expression of a first and second cistron. The first citrus encodes a secretion signal sequence (malEou stll), a light chain constant and variable domain, and a gD marker. The second citron encodes a secretion signal sequence, a sequence that encodes a constant and variable heavy chain domain (CH1) domain and a cysteine dimerization domain and at least a portion of the viral dacapsid protein.

Figure 6 shows the sequences of the huMAb4D5-8 light and heavy chain structural region. Superscript / bold numbers indicate amino acid positions according to Kabat numbering.

Figure 7 shows modifications / variations in the huMAb4D5-8 light and heavy chain structural region sequences. The numbers in superscript / bold indicate the amino acid positions according to Kabat numbering.

Figure 8 illustrates the randomization scheme for each diversified CDR position in the YSGR-A, YSGR-B, YSGR-C, and YSGR-D libraries as described in Example 1.

Figures 9A-9D show the mutagenic oligonucleotides used in the construction of the YSGR-A, YSGR-B, YSGR-C, YSGR-D libraries as described in Example 3. Equimolar weakened DNA are represented in the codon set (W = A / T , K = G / T, M = A / C, N = A / C / G / T, R = A / G, S = G / C, Y = T / C). The codon set is represented in the codeRJB. The notation "XXX" in H3-A6-H3-A17 oligonucleotides represents Tyr / Ser / Gly coding codons in a molar ratio of 50/25/25, respectively. The notation "XXX" in the H3-B6-H3-B17 oligonucleotides represents Tyr / Ser / Arg coding codons at a molar ratio of 25/50/25, respectively. The notation "XXX" in the H3-C6-H3-C17 oligonucleotides represents codons coding for Tyr / Ser / Gly / Arg at a molar ratio 38/25/25/12, respectively. The notation "XXX" in oligonucleotides H3-D6-H3 to D17 represents coding codes for Tyr / Ser / Gly / Arg / Ala / Asp / Glu / Phe / His / lle / Lys / Leu / Met / Asn / Pro / GIn / ThrA / al / They have a molar ratio of 20/26/26/13/1/1/1/1/1/1/1/1/1/1/1/1/1/1, respectively.

Figure 10 shows the enrichment ratio for the MySGR-AD library after 5 selection steps against human DR5 or human HER-2 as described in Example 2. Numbers are shown as X / Y, where X represents the number of clones. Y are the number of clones that specifically bind to human DR5 or human HER-2. Specific clones are identified as those that have at least ten-fold greater binding to human DR5 or human HER-2 (based on ELISA signal reading at 450nm) than binding to bovine serum albuminate (BSA).

Figure 11A shows the CDRH1, CDRH2, CDRH3 and CDRL3 sequences for 106 clones that bind to human HER-2. Figure 11B shows ELISA assay results for each of the clones set forth in Figure 11A. Bold numbers indicate a strong connection (sign 2-10).

Figure 12 shows the amino acid sequences for CDRL3, CDRH1, CDRH2, and CDRH3 of human HER-2-specific ligands with short isolated regions (e.g., 6-7 residues) of CDRH3 from the library GSGR-AD as described in Example 2. Consensus sequences are displayed for CDRL3, CDRH1, and CDRH2. (Clone numbers correspond to those shown in Figure 11.)

Figure 13 shows the amino acid sequences for CDRH1, CDRH2, and CDRH3 of human HER-2-specific ligands with a CDRH3 having 8 amino acids isolated from the YSGR-AD library as described in Example 2. The consensus sequences are shown for CDRH1, CDRH2 and CDRH3. (Clone numbers 1-19 correspond to those shown in Figure 11 as follows: 17, 97, 18, 19, 98, 99, 100, 20, 21,22, 23, 24, 101, 102, 25, 103, 26, 27 and 28, respectively.)

Figure 14 shows amino acid sequences for CDRH1, CDRH2 and CDRH3 from human HER-2 specific ligands with CDRH3 medium length regions (e.g., about 12-14 amino acids) isolated from YSGR-AD libraries as described in the Example 2 . The consensus sequences are displayed for CDRH1, CDRH2, and CDRH3. Consensus sequences are displayed for CDRH3 by transferring some of the CDRH3 sequences along two amino acids, so that the CDRH3 sequence starts at position 97 instead of position 95.

Figure 15 shows the CDRL3, CDRH1, CDRH2 and CDRH3 sequences of human DR5 ligands, and the IC50 of some human DR5 ligands. Figure 16 shows the amino acid sequences CDRL3, CDRH1, CDRH2 and CDRH3. Human DR5 is isolated from the YSGR-AD library as described in Example 2. The IC50 of clones for binding to human DR5 are displayed. Clones that cross react with murine DR5 are also identified. The consensus sequences are displayed for CDRL3, CDRH1, CDRH2, and CDRH3. (Clone numbers 1-11 correspond to clone numbers 10, 11, 12, 8, 7, 13, 5, 9, 6, 15, and 14 in Figure 15.)

Figure 17 shows the binding curve of human DR5-specific ligands isolated from the YSGR-A-D library. Some of the specific ligands also bind to murine DR-5.

Figure 18 shows a 3D model illustrating Apo-2L ligand, Antibody and BFD1 antibody bind to the DR5 receptor. The antibody binding region overlaps each other. The binding site of these antibodies is distinct from most residues of the Apo-2L ligand binding site.

Figures 19A and 19B illustrate the diversified CDR position randomization scheme in the H3 Binary libraries (SAH3, SCH3, SFH3, SGH3, SIH3, SLH3, SPH3, SRH3, STH3, SWH3 and SYH3), as described in Example 4. Indicated amino acid positions are numbered according to the Kabat numbering.

Figures 20A-20L show the mutagenic oligonucleotides used in the construction of the binary libraries H3 (SAH3 (Fig. 20A), SCH3 (Fig. 20B), SFH3 (Fig. 20C), SGH3 (Fig. 20D), SIH3 (Fig. 20E). ), SLH3 (Fig.20F), SNH3 (Fig. 20G), SPH3 (Fig. 20H), SRH3 (Fig. 201), STH3 (Fig. 20J), SWH3 (Fig. 20K), and SYH3 (Fig. 20L) )), As described in Example 4 (SEQ IDNs: 618-788). Equimolar degeneration of DNA is represented in codon sets (W = A / T, K = G / T, M = A / C, N = A / C / G / T, R = A / G, S = G / C , Y = T / C). Codon sets are represented in code IUB.Figure 21A shows the amino acid sequences CDRL3, CDRH1, CDRH2, and CDRH3 of HER2-specific ligands isolated from the binary library pool H3 (SXH3), as described in Example 5 (SEQ ID Nos: 789-896). Figure 21B shows the ELISA test results for each of the clones set out in Figure 21A. Dark shading indicates a strong binding character (2 to 10 signal).

Figure 22A illustrates the randomization scheme for diversified CDR decapitation in the Surface Binary libraries (SY, SW andSF) as described in Example 6. The indicated amino acid positions are numbered according to Kabat numbering.

Figure 23 shows the mutagenic oligonucleotides used in the construction of surface Binary libraries (SY, SW and SF) as described in Example 6 (SEQ ID Nos: 1005-1013). Equimolar degeneration of DNA is represented in codon sets (W = A / T, K = G / T, M = A / C, N = A / C / G / T, R = A / G, S = G / C , Y = T / C). Codon sets are represented in the IUB code.

Figure 24A shows the amino acid sequences CDRL3, CDRH1, CDRH2, and CDRH3 from HER2-specific ligands isolated from a surface binary library (SX-surface), as described in Example 8 (SEQ ID Nos: 897-1004). Figure 24B shows the ELISA test results for each of the clones illustrated in Figure 24A. Dark shading indicates a strong binding character (2 to 10 signal), light shading indicates poor binding (0.25 to 2 signal).

Figure 25 graphically represents the specificity of Fabs containing different binary amino acid combinations (SenTyr, Ser: Trp, SenArg or SenPhe) obtained at present from the SXH3 binary library or SX-surface binary library.

Figures 26A and 26B show plasmon surface resonance analyzes of binding from Fab soluble proteins of three HER2 ligand clones (Clone B1L, clone G54 and clone YSGR-A-42) to immobilized aHER2. Clone B1L had a ka of 1.9 x 106 M "1 S" 1, a kdde of 1.7 x 10 "3 S" 1 and an KD of 890 nM. Clone G54 had a ka of 2.0 x 105 M "1 S" 1, a kd of 2.2 x 10 "3 S ~ 1 and a Kd of 11 nM. Clone YSGR-A-42 had a ka of 2 , 7 x 10 6 M-1S "1, a 1.5 x 10" 3 s Kd and a 570 pM KD.

Figure 27 shows the results of flow-through cytometric analysis of binding of anti-HER2 Fabs isolated from each of the YESR (clone A-42), SX-surface (clones G37 and G54), and SXH3 (clone B1L) libraries for NR6 cells. or H2NR6-4D5 as described in Example 7.

Figure 28 shows the sequences for CDRH1, CDRH2, CDRH3 and CDRL3 for each of the HER2 binding IgGs B1I, G37, G54, YSGR-A-42, YSGR-A-27, B27, G43 and YSGR-D-104. Figure 28 also shows IC50 values for the Fab version of each clone.

Figure 29 shows the results of the competitive binding assays described in Example 7 to determine the ability of each of the indicated HER-2 specific IgGs to compete for HER2 binding with Omnitarg, Herceptin and each of the other IgGs.

Modes of Conduct of the Invention

The invention provides novel, unconventional, many simplified and flexible methods for diversifying CDR sequences (including antibody variable domain sequences) and libraries comprising a multiplicity and generally a large multiplicity of diverse CDRs (including antibody variable domain sequences). Such libraries provide combinatorial libraries useful for, for example, selecting and / or screening synthetic antibody clones with desirable activities such as binding affinity and avity affinity. These libraries are useful for identifying immunoglobulin polypeptide sequences that are capable of interacting with any of a wide range of target antigens. For example, many libraries comprising the immunoglobulin polypeptides of the invention, expressed on display by phage, are particularly useful for, and provide for high, efficient production and automated systems for screening and / or screening for antigen-binding molecules of interest. The methods of the invention are designed to provide high affinity ligands to target antigens with minimal changes to a source or model molecule and provide good yield when the antibody or antigen-binding fragment is produced in cell culture.

The methods and compositions of the invention provide numerous additional advantages. For example, relatively simple CDR variant sequences can be generated using a codon set that encodes a restricted number of amino acids (as opposed to the conventional approach of using a codon set to encode the maximum number of amino acids), while maintaining sufficient diversity of one codon. single target binding frequency. The simplified (and generally relatively smaller) nature of the sequences generated according to the invention allows for further diversification into a population or subpopulation of this, which has been identified as having the desired characteristics.

The simplified nature of the target antigen ligand sequences obtained by the methods of the invention leaves a significantly larger space for further sequence modifications and to achieve the desired results. For example, the sequence of these modifications is routinely performed by affinity maturation, humanization, and the like. By diversifying based on a restricted decoding set that encodes only a limited number of amino acids, it becomes possible to achieve different epitopes using different restricted set sets, thus providing the subject matter technician with greater control of diversification, as compared to randomization-based procedures. a maximum number of amino acids. The advantage of using a restricted codon set is that undesirable amino acids can be deleted, for example methionine or codons encountered, thus improving the overall quality and productivity of the library. In addition, in some cases it may be desirable to limit the ligands' diversity of binders. potentials. Methods and compositions of the invention provide the flexibility necessary to achieve this goal. For example, the presence of certain amino acids, such as tyrosine, in a sequence results in fewer rotational conformations.

Definitions

Amino acids are represented herein either by the one letter code or by the three letter code or both.

The term "affinity purification" describes the purification of a molecule based on the specific attraction or binding of the molecule to a chemical or binding partner to form a combination or complex that allows the molecule to be separated from impurities while remaining attached or attracted to the molecule. Link.

The term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (including eagonist antagonist antibodies), polyepitopic specificity antibody compositions, affinity matured antibodies, humanized antibodies, chimeric antibodies as well as antigen-binding fragments, for example Fab, F (ab ') 2, scFv and Fv, provided they exhibit the desired biological activity. In one example, the term "antibodies" also includes human antibodies. As used herein, "antibody variable domain" refers to the light and heavy chain moieties of antibody molecules that include amino acid sequences from non-complementarity determining regions (CDRs, for example, CDR1, CDR2 and CDR3), and regions called regions. Structural Funds (FR). Vh refers to the heavy chain variable domain. Saw. refers to the light chain variable domain. According to the compositions and methods used in the present invention, the amino acid positions assigned to the CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, 1987 and 1991)). The amino acid numbering of antibodies or antigen-binding antibody fragments is also numbered according to Kabat numbering.

As used herein, the term "non-complementarity determining region" (CDRs; i.e. CDR1, CDR2, and CDR3) refers to the presence of amino acid residues an antibody variable domain that are required for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region may consist of amino acid residues of a "complementarity determining region" as defined by Kabat (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain. light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain: Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. National Institutes of Health, Bethesda, MD 1991) and / or "hypervariable loop" residues (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol.196: 901-917 (1987)). In some cases, a complementarity determining region may include amino acids the defined CDR region according to Kabat and a hypervariable loop. For example, the 4D5 antibody heavy chain CDRH1 includes amino acids 26 to 35. The consensus sequence for CDRL1 (as defined by Kabat) in the 4D5 antibody is R-A-S-Q-D-V-N-T-A-V-A (SEQ ID NO: 29). The consensus sequence for CDRL2 (according to the definition of Kabat) in antibody 4D5 is S-A-S-L-Y-S (SEQ ID No: 30).

"Structural region" (hereinafter "FR") are variable domain residues that are not CDR residues. Each variable domain usually has four FRs identified as FR1, FR2, FR3, and FR4. If CDRs are defined according to Kabat, FR light chain residues are positioned about residues 1-23 (LCFR1), 35 ^ 9 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4) and FR heavy chain residues are positioned at about residues 1-30 (HCFR1), 36.9 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4). In some cases, when CDRs comprise amino acids from both the Kabat-defined CDRs and the hypervariable loop, the FR residues may be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, FR1 heavy chain residues are at positions 1-25 and FR2 residues at positions 36-49.

As used herein, "codon set" refers to a set of different sequences of three nucleotides used to encode the desired amino acid variant. A set of oligonucleotides may be synthesized, for example, by solid phase synthesis, including sequences representing all possible combinations of nucleotide triads provided by the codon set that will encode the desired amino acid group. A standard form of codon designation is that of the code EJB, which is known in the art and described herein. A codon set is usually represented by 3 issalic capital letters, for example, NNK, A / A / S, XYZ, DVK, and the like. Synthesis of oligonucleotides with the "degeneracy" of selected nucleotides at certain positions is well known in the art, for example, the TRIM approach (Knappek et al .; J. Mol. Biol. (1999), 296: 57-86); Garrard & Henner, Gene (1993), 128: 103). Such sets of oligonucleotides with certain codon sets may be synthesized using commercial nucleic acid synthesizers (available from, for example,

Applied Biosystems, Foster City, California), or may be obtained commercially (for example, from Life Technologies, Rockville, MD). Therefore, a set of synthesized oligonucleotides having a specific codon set will usually include a variety of different sequence oligonucleotides, the differences established by the codon set of the overall sequence. Oligonucleotides, as used in the present invention, have sequences that allow hybridization of a variable domain nucleic acid template and may also, but not necessarily, include sites useful for the use of restriction enzymes, for example for cloning purposes.

The term "restricted codon set", and variations thereof, as used herein refer to a codon set encoding a much more limited number of amino acids than codon sets commonly used in diversity producing methods known in the art. In one aspect of the invention, the restricted decoding sets used to encode sequence diversity from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or just 2 amino acids. In some embodiment examples, a restricted codon set used for sequence diversification encodes 2, but at least 10 or less, 8 or less, 6 or less, 4 or less amino acids. In a typical example, a tetranomial decode set is used. Examples include codon tetranomial assemblies including RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, and WMT, as are known in the art. In another characteristic example, binomial codon sets are used. Examples of binomial decode sets include TMT, KAT, YAC, WAC, TWC, TYT, YTC, WTC, KTT, YCT, MCG, SCG, MGC, SGT, TAB, GKT, and GYT. Determination of suitable codon constraints, as well as specific identification of amino acids encoded by a specific restricted codon, is well known and apparent to one skilled in the art. Determinations of suitable amino acid sets to be used for diversification of a CDR sequence may be empirical and / or guided by criteria known in the art (e.g., the inclusion of a combination of hydrophobic and hydrophilic amino acid types, etc.).

An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of one heavy chain and one light chain variable domain dimer in tight association which may be covalent in nature, for example, noscFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the VH-VL dodimer surface. Collectively, the six hypervariable regions confer antigen binding specificity to the antibody. However, even an isolated variable domain (or half of an Fv that comprises only three antigen-specific hypervariable regions) has the ability to recognize and bind the antigen, albeit at lower affinity than the complete deletion site.

The "Fab" fragment contains the light chain constant and variable domain and the first heavy chain constant domain (CH1). F (ab ') 2 antibody fragments consist of pairs of Fab' fragments that are usually covalently linked near their carboxy-terminal region by the cisterns of the hinge region therebetween. Other chemical couplings of antibody fragments are also known in the prior art.

Fragments of the "single stranded Fv" or "scFv" antibodies comprise the Vh and Vl domains of the antibody, in which these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and V1 domains that allows the scFv to form the desired antigen binding structure. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore Eds., Springer-Verlag, New York, p. 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen binding sites, which comprise a variable domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (Vh and Vl). Using a ligand that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are more fully described, for example, in EP 404,097, WO 1993/11161 and in Holinger et al., Proc. Natl. Acad. Know. USA, 90: 6444-6448 (1993).

The term "linear antibodies" refers to antibodies described in Zapata et al., Protein Eng., 8 (10): 1057-1062 (1995). Briefly, these antibodies include a pair of tandem Fv segments (Vh-Ch1-Vh -Ch1), which together with the complementary stranded polypeptides form a pair of antigen binding regions. Linear antibodies may be mono or bispecific.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present. in smaller quantities. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike polyclonal antibody preparations which include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single antigen determinant. The adjective "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be considered as requiring antibody production by any particular method. Monoclonal antibodies to be used according to the present invention may be prepared, for example, by the hybridoma method described first by Kohler et al, Nature, 256: 495 (1975), or may be made by recombinant DNA methods. (see, for example, US Patent 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

Monoclonal antibodies of the present invention specifically include "chimeric" antibodies (immunoglobulins), wherein a portion of the light and / or heavy chain is identical or homologous to the corresponding antibody sequences derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the identical or homologous chain (s) corresponding antibody sequences derived from other species or belonging to another class or subclass of antibodies, as well as fragments thereof, provided they exhibit the desired biological activity (US Patent 4,816,567; and Morrison et al., Proc. Natl. Acad.Sei. USA, 81: 6851-6855 (1984)).

"Humanized" forms of non-human antibodies (e.g., murine) are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody), where residues from a patient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate that has the specificity, affinity and capacity desired. In some cases, the framework immuno-framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all regions. These are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of the immunoglobulin (Fc) constant region, typically that of human immunoglobulin.For further details, see Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323 -329 (1988); and Presta, Curr. Op. Struct. Biol.2: 593-596 (1992).

"Species-dependent antibody" is one that has a binding strength for an antigen of a first mammalian species than for a homologous antigen of a second mammalian species. Typically, species-dependent antibodies "specifically bind" to an antigen (i.e. it has, for example, a binding affinity (Kd) of not more than 1 x 10 ~ 7 M, not more than 1 x 10 - 8 M, and for example not more than 1 x 10 - 9 M ), but has an affinity for a homologous antigen of a second non-human mammal species that is at least about 50 times, or at least 500 times, or at least 1000 times, weaker than its affinity. binding to the human antigen. Species-dependent antibodies may be any one of several types of antibodies as defined above, but is preferably a humanized or human antibody.

As used herein, "mutant antibody" or "anti-variant" refers to a variant amino acid sequence of species-dependent antibodies in which one or more of the amino acid residues of species-dependent antibodies have been altered. Such mutants necessarily have less than 100% sequence identity or similarity to species dependent antibodies. In one example, the mutant antibody will have an amino acid sequence that has at least 75% amino acid sequence identity or amino acid similarity to both the heavy chain variable domain and the light chain variable domain of the species-dependent antibodies, for example at least 80%, for example at least 85%, for example at least 90% and for example at least 95%. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (ie same residue) or similar (ie amino acid residues from the same group based on common side chain properties, see below) with species-dependent antibody residues after sequence alignment and introduction of degaps, if necessary, to achieve the maximum percent identity in the sequence. No N-terminal, C-terminal, internal extensions, deletions, or insertions into the antibody sequence outside the variable domain should be interpreted as a sequence that affects identity or similarity.

An "isolated" antibody is one that has been identified, separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to more than 95 wt% of the antibody as determined by the Lowry method, and more preferably more than 99 wt%; (2) to a degree sufficient to obtain at least fifteen spinning cup residues; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Isolated antibody includes the antibody in situ in recombinant cells, provided that at least one component of the antibody's natural environment is not present.

Normally, however, the isolated antibody will be prepared by at least one purification step.

The term "antagonist" is used in the broad sense of the word and includes any molecule that completely or partially blocks, inhibits or neutralizes one or more biological activity of the target antigen described in the present (e.g. DR5 and HER-2) in vitro, in or out of order. alive. Examples of such DR5 biological activities include Apo2L / TRAIL binding to DR5, induction of apoptosis, as well as new activities reported in the literature. Examples of such HER-2 biological activities include binding of ligands such as heregulin, phosphorylation. HER-2 tyrosine induction, proliferation induction as well as apoptosis induction as well as new activities reported in the literature. An antagonist can function directly or indirectly. For example, the antagonist may function to partially block, inhibit or neutralize one or more biological activities of a target molecule ligand, in vitro, in situ, or in vivo, as a result of its direct binding to the target molecule. The antagonist may also have an indirect function to partially or totally block, inhibit or neutralize one or more biological activities of the target molecule, in vitro, in situ, or in vivo, as a result of, for example, blocking or inhibiting another effector molecule. Antagonist molecule may include a "double" antagonist activity in which the molecule is capable of blocking partially or totally, inhibiting or neutralizing a biological activity of the target molecule.

The term "agonist" is used in the broad sense of the word and includes any molecule that enhances, stimulates or fully or partially activates one or more biological activity of the target antigen described herein (for example, DR5 and HER-2) in vitro, in situ or in vitro. alive. Examples of such DR5 biological activities include Apo2L binding and apoptosis, as well as new activities reported in the literature. Examples of such biological activities of HER-2 include ligand binding such as heregulins, receptor tyrosine phosphorylation inducing proliferation, apoptosis, as well as new activities reported in the literature. An agonist can function directly or indirectly. For example, the agonist may have the function of partially or fully enhancing, stimulating or activating one or more biological activities of a ligand of a target molecule, either in vitro, in situ, or inventively, as a result of its direct binding to the target molecule that causes receptor activation or signal transduction. The agonist may also have indirect function to partially or fully enhance, stimulate or activate one or more biological activities of the target molecule, in vitro, in situ, or in vivo, for example by stimulating another effector molecule that will culminate in target molecule activation or signal transduction. It is contemplated that an agonist may act as an enhancer that functions indirectly to enhance or increase the activation or activity of the target molecule. For example, an agonist may improve endogenous Apo-2L activity in a mammal. This could be achieved, for example, by pre-complexing DR5 or stabilizing complexes of the respective DR5 receptor ligands.

"Cell", "cell line" and "cell culture" are used interchangeably and these names include all progenies of a cell or cell culture. Thus, the terms "transformed" and "transformed cells" (or transformed cells) include the primary cell and its derived cultures without regard to the amount of transfers.

It is also understood that all offspring may not be precisely identical in the context of DNA due to deliberate or inadvertent mutations. Mutant progenies that have the same biological function or activity selected for the originally modified cell are included. When different designations are intended, this will be clear from the context.

The term "control sequences" means DNA sequences necessary for the expression of a coding sequence operably linked in a specific host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome deletion site, and possibly other poorly understood sequences. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers.

The term "capsid protein" refers to a protein, or at least a part of which is present on the surface of the virus particle. From a functional perspective, a capsid protein is any protein, which is associated with a virus particle during the viral assembly process in the host cell, and remains associated with the assembled virus until it infects another host cell. The capsid protein may be the major capsid protein (major) or may be the minor capsid protein (minor). The major major protein is generally a capsid protein that is present in the viral capsid in at least about 5, at least 7, or at least 10 copies of the proteins or more. The main viral capsid protein can be present in tens, hundreds or even thousands of copies per virion. An example of a major capsid protein is filamentous phage protein P8.

The "limit of detection" of a chemical entity in a given assay is the minimum concentration of the entity that can be detected above the background level for this analysis. For example, in phage ELISA the "detection limit" for a particular display of a phage-specific antigen-binding fragment is the concentration of phage in which the specific phage produces an ELISA signal below that produced by a control phage that does not display the phage-specific phage. antigen binding.

"DR5 Receptor" or "DR5" when used herein encompasses native receptor sequences and variant receptor sequences. These include DR5 receptors expressed in a variety of mammals, including humans. The DR5 receptor may be endogenously expressed as it occurs naturally in a variety of human tissue strains, or may be expressed by synthetic or recombinant methods. A "native DR5 receptor sequence" comprises a polypeptide with the same amino acid sequence as a naturally derived DR5 receptor. Thus, a native DR5 receptor sequence may have the naturally occurring sequence of amino acids at the DR5 receptor of any mammal. Such native DR5 receptor sequences may be isolated from nature or may be produced by synthetic or recombinant means. The term "native DR5 receptor sequence" specifically encompasses naturally occurring secreted or deleted forms (e.g., a soluble form containing, for example, an extracellular domain sequence), naturally occurring variant forms (e.g., co-alternating forms) and variants natural allelics. Receptor variants may include fragments or mutants by deletion of native DR5 receptor sequences. The 411 amino acid sequence of human DR5 is shown in Table 1 and is the sequence of Figure 3A, as published in WO98 / 51793 on November 19, 1998. Transcriptional splicing of human DR5 variant is known in the state of the art. This variant DR5 splice encodes the 440 amino acid sequence of human DR5 shown in Figures 3B and 3C as published in WO 98/35986 on August 20, 1998. Murine DR5 polypeptide sequences and an DR5 extracellular domain are also shown in Table 1. below, down, beneath, underneath, downwards, downhill.

DR5 biological activities include (a) ability to induce or stimulate or signal apoptosis in at least one type of mammalian cancer cell or virus infected cells in vivo or ex vivo, (b) ability to bind to Apo2L / Naturally occurring TRAIL. Assays to determine biological activity such as apoptosis can be performed by methods known in the art such as DNA fragmentation (see, for example, Marsters et al., Curr. Biology, 6: 1669 (1996)) , caspase inactivation, DR5 binding (see, for example, WO98 / 51793, published November 19, 1998. The terms "apoptosis" and "apoptotic activity" are used broadly, and refer to orderly or controlled cell death in mammals that is usually accompanied by one or more characteristic cell events, including cytoplasm condensation, loss of plasma membrane microvilli, nucleus segmentation, D degradation Chromosomal NA or loss of mitochondrial function. This activity can be determined by measurement, for example, by cell viability assays (such as Alamar blue staining or MTT assays), FACS analysis, caspase activation, DNA fragmentation (see, for example, Nicoletti et al., J Immunol Methods, 139: 271-279 (1991), and poly-ADP ribose polymerase, "PARP", cleavage assay known in the art.

"DR5 receptor antibody", "DR5 antibodies", or "anti-DR5 antibodies" are used in a broad sense to refer to antibodies that bind to at least one form of a DR5 receptor. Optionally the DR5 antibody is fused to or linked to a heterologous sequence or molecule. Preferably the heterologous sequence allows or assists the antibody to form oligomeric or higher order complexes.

Optionally, the DR5 antibody binds to DR5 receptors but does not cross-react or cross-react with any additional Apo-2L receptor (e.g., RP4, DcRI, or DcR2). Optionally, the antibody is a signaling agonist for DR5 activity. Optionally, the DR5 antibody of the invention binds to a DR5 receptor at a concentration between 0.1 nM to 20 mM as measured in a BlAcore binding assay (as described herein). Optionally, some embodiments, the antibodies of the invention display. an IC 50 value of from about 1 nM to about 20 nM as measured in a binding assay (such as phage ELISA, as described in the examples below).

The terms "Apo2l_ / TRAIL", "Apo-2L", and "TRAIL" are used herein to refer to a polypeptide sequence which includes amino acid residues 114-281, including residues, 95-281, including residues, 92-281, including residues, 91-281, including residues, 41-281, including residues, 15-281, including residues, 1-281, of the amino acid sequence shown in Table 1, as well as biologically active fragments, variants with deletions, insertions or substitutions of the sequences referred to above. Apo-2L polypeptides may be encoded by native nucleotide sequences as described and shown in Figure 1 of WO2005100399.

A "fusion protein" and "fusion polypeptide" refer to a polypeptide having two portions covalently linked together, where each portion is a polypeptide having a different property. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple physical or chemical property, such as binding to a target antigen, a catalyst reaction, and the like. The two moieties may be directly linked by a single peptide bond or via a linker peptide containing one or more amino acid residues. Generally, the two portions and the binder will be in the reading frame together. In certain embodiments, the two polypeptide moieties are either obtained from different or heterologous polypeptides.

"Heterologous DNA" is any DNA that is introduced into a host cell. DNA can be derived from a variety of sources, including genomic DNA, cDNA, synthetic DNA, and fusions or combinations thereof. DNA may include DNA from the same cell or cell type as host or recipient cells or DNA from different cell types, for example from a mammal or plant. The DNA may optionally include a selectable or selectable gene, for example antibiotic resistance genes, temperature resistance genes, and the like.

As used herein, "highly diverse position" refers to the position of an amino acid located in the light and heavy variable regions that has a different number of amino acids in the position compared to known and / or antigen-binding amino acid sequences or fragments. natural occurrence. Highly diversified positions are generally in the CDR regions. In one aspect, the ability to determine highly diverse positions in known and / or naturally occurring antibodies is facilitated by data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md, 1987 and 1991). An Internet-based database located at http:: www.bioinf.org.uk.abs.structures.html offers an extensive collection and alignment of light chain sequences (http:: www.biomf.org.uk.abs). .lc.aliqn) and heavy chain (http //: www.bioinf.orq.uk.abs.hc.aliqn) and facilitates the determination of highly diversified positions in these sequences. According to the invention, an amino acid composition is highly diverse if it has from about 2 to 11, from 4 to 9, and / or about 5 to 7 possible different variations in amino acid residue positions. In some embodiments, an amino acid position is highly diverse if it has at least about 2, at least about 4, at least about 6, and / or at least about 8 possible different variations in the positions of the residues. amino acids.

As used herein, "library" refers to a variety of antibody sequences or antibody fragments (e.g., the polypeptides of the invention), or nucleic acids encoding such sequences, which are different sequences in the variant combination of amino acids that are introduced into these sequences according to the methods of the invention.

"Bonding" is the process of forming phosphodiester bonds between the two nucleic acid fragments. For the binding of the two fragments, the ends of the fragments must be compatible with each other.

In some cases, the extremities will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered end commonly produced after digestion with endonucleases to a blind end and make it compatible for allocation. To produce a blunt end, the DNA is treated in a suitable buffer for at least 15 minutes at 15 ° C with about 10 Klenow DNA polymerase I or T4 DNA polymerase units in the four deoxyribonucleotide triphosphates present. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation or silica purification.

The DNA fragments that will be ligated are placed in a solution with equimolar quantities. The solution will also contain ATP, ligase buffer and ligase such as T4 DNA ligase, about 10 units per 0.5 µg DNA. If DNA is or is bound in a vector, the vector is first linearized by digestion with appropriate restriction endonuclease (s). The linear fragment is then treated with bacterial alkaline phosphatase or bovine intestinal alkaline phosphatase to prevent self-ligation during the ligation step. Other methods are well known in the state of the art.

"Mutation" is a deletion, insertion, or substitution of a nucleotide (s) with respect to a reference nucleotide sequence such as a wild-type sequence.

The term "natural" or "naturally" antibody as used herein refers to antibodies identified from a non-synthetic source, for example, from an ex vivo specific differentiated B-cell antigen, or its lineage. corresponding cellular hybridoma, or from antibodies obtained from the serum of an animal. These antibodies may include antibodies raised in any type of immune response, whether natural or otherwise induced. Natural antibodies include amino acid sequences, and nucleotide sequences that constitute or encode these antibodies, for example, as identified in the Kabat database. As used herein, natural antibodies are different from "synthetic antibodies", synthetic antibodies refer to antibody sequences that have been altered from a source sequence or by model, for example, by substituting, deleting or adding an amino acid, or more. of an amino acid at a given position with a different amino acid, the different amino acids provide a different antibody antibody sequence to the source antibody sequence.

"Operationally linked" refers to a nucleic acid means that the nucleic acid is placed in functional relationship with another nucleic acid sequence. DNA for a prior sequence or secretion leader, for example, is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the polypeptide secretion; an enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if positioned to enable translation. Generally, "operably linked" indicates that the linked DNA sequences are contiguous and, in the case of the secretion leader, contiguous and in the reading frame. The enhancers, however, need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If these sites do not exist, oligonucleotide adapters or ligands are used in accordance with conventional practice.

"Phage display" is a technique in which variant polypeptides are displayed as fusion proteins to at least a portion of the capsid protein on the surface of the phage, e.g. filamentous phage, particles. The usefulness of phage display lies in the fact that large libraries of randomized variant proteins can be quickly and effectively classified for those sequences that bind to a high affinity target antigen. Exposure of the peptide and phage protein library has been used to screen millions of polypeptides to select those with specific binding properties. Polyvalent phage display methods were used to display small-reacted peptides and small proteins by fusing both gene III and filamentous phage gene VIII. Wells and Lowman, Curr. Opin.Struct. Biol, 3: 355-362 (1992), and the references cited therein. In a monovalent phage display, a protein or peptide library is fused to a common gene III or part thereof, and expressed at low levels in the presence of the wild type protein III genus so that the phage particles exhibit a copy or none of the fusion proteins. . The effects of avity are relatively reduced on the polyvalent phage so that classification is based on intrinsic ligand affinity, and phagemid vectors are used, which will simplify DNA manipulations. Lowman and Wells, Methods: Accompaniment to Methods in Enzymology, 3: 205-0216 (1991).

A "phagemid" is a vector plasmid having a bacterial origin replication, for example ColE1, and a copy of an intergenic region of a bacteriophage. The phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage. The plasmid will usually also contain a selectable marker for antibiotic resistance. Cloned DNA segments in these vectors can be propagated as plasmids. When cells containing the vectors are provided with all genes necessary for phage particle production, the plasmid replication mode alters the circle of replication to generate copies of a strain of plasmid DNA and phage envelope particles. The phagomid may form infectious and non-infectious phage particles. This term includes phagemids that contain a phage capsid protein gene or fragment thereof linked to a heterologous polypeptide as a fusion gene such that the heterologous polypeptide is displayed on the phage surface particle.

The term "phage vector" means a replicative form of a double-stranded bacteriophage containing a heterologous gene capable of replication. Phage ovector has the origin of phage replication allowing replication and deformation of the phage particle. In certain embodiments, the phage is a filamentous bacteriophage such as phage M13, fl, fd, Pf3 or a derivative thereof, or a lambdoid phage such as lambda, 21, phi80, phi81, 82, 424, 434 and etc. , or a derivative thereof.

"Oligonucleotides" are short-stranded, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite or phosphoramidite chemists using solid phase techniques as described in EP 266032 published May 4, 1988, or by deoxynucleoside H-phosphonate as described by Froeshler et al., Nucl. Acids Res., 14: 5399-5407 (1986)).

Additional methods include polymerase chain reaction defined below and other self priming methods and syntheses of oligonucleotides on solid supports. All of these methods are described in Engels et al., Agnew. Chem.Int. Ed. Engl. 28: 716-734 (1989). These methods are used if the full nucleic acid sequence of the genes is known, or if the complementary nucleic acid sequence coding strand is available.

Alternatively, if the target amino acid sequence is known, potential nucleic acid sequences can be inferred using known and preferred decoding residues for each amino acid residue. Soligonucleotides may be purified on polyacrylamide gel or columns of molecular size or by precipitation.

DNA is "purified" when DNA is separated from impurities that are not nucleic acids. Impurities can be polar, nonpolar, ionic and so on.

A "source antibody" as used herein refers to an antibody or fragment that binds to an antigen whose sequence serves as a template sequence upon which diversification according to the criteria described herein is performed. In certain embodiments, an antigen-binding sequence generally includes an antibody variable region, and at least one CDR including the structural region.

The term "solvent accessible position" as used herein refers to a position of an amino acid residue in the light and heavy chain variable regions of a source antibody or antigen-binding fragment which is determined, based on structure, grouped together. structure and / or modeled on the structure of the antibody or antigen-fragmenting, as potentially available for solvent access and / or by contacting a molecule, such as an antibody-specific antigen. These positions are usually found on the CDRs and on the protein exterior. The solvent accessible positions of an antibody or antigen-binding fragment as defined herein may be determined using a number of known algorithms in the art state. In certain embodiments, the solvent accessible position is determined using coordinates of three-dimensional models of an antibody (or part thereof, for example, an antibody variable domain, or CDR segment (s)), using a computer program such as Insight II program (Accelrys, San Diego, California). Solvent-accessible positions can also be determined using state-of-the-art algorithms (e.g., Lee and Richards, J. Mol. Biol. 55, 379 (1971) and Connolly, J.Appl. Cryst. 16, 548 (1983)). Determination of solvent accessible positions can be performed using suitable software for modeling and obtaining the three-dimensional structural information of the protein from an antibody (or portion thereof). The software that can be used for these purposes is SYBYL Biopolymer Module software (Tripos Associates). Generally, in certain embodiments, where an algorithm (program) requires the use of the size parameter input, the "size" of a probe which is used in the calculation is set at about 1.4 Angstrom or less. In addition, the determination of solvent-accessible regions using area methods using personal computer software has been described by Pacios ((1994) "ARVOMOUCONTOUR: molecular surface areas and volumes on Personal Computers." Comput. Chem. 18 (4): 377 -386; and (1995). "Variations of Surface Areas and Volumes in Distinct Molecular Surfaces of Biomolecules." J. Mol. Model. 1: 46-53.)

A "transcriptional regulatory element" will contain one or more of the following elements: an enhancer element, a promoter, an operator sequence, a repressor gene, and a transcription stop sequence. These components are well known in the art. US Patent 5,667,780.

A "transformed" cell is a cell that receives and maintains DNA as evidenced by the expression of a phenotype associated with DNA (for example, antibiotic resistance conferred by a DNA-encoded protein).

"Modification" refers to a process by which a cell receives DNA becomes a "transformed" cell. DNA uptake can be permanent or transient.

A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or was made using any of the human antibody production techniques as disclosed herein. This definition of human antibody specifically excludes a humanized non-human antigen-binding residue account antibody.

"Affinity matured" antibody is one with one or more changes in one or more of its CDRs, which results in increased antibody affinity for antigen, compared to parental antibody that does not have this change (s). In preferred embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio / Technology, 10: 779-783 (1992) describes affinity maturation through VH and VL domain exchange. Random mutagenesis of structure and / or CDR residues is described by: Barbas et al., Proc. Natl. Acad. Sci., U.S.A.91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154 (7): 3310-9 (1995); and Hawkins et al., J. Mol. Bioi, 226: 889-896 (1992).

The "Kd" or "Kd value" is the dissociation constant for the interaction of one molecule with another. In one example, the Kd value is measured by a radiolabeled protein binding (RAI) test.

In one embodiment, an RIA assay for DR5 or HER-2 may be performed with the Fab version of an anti-DR5 or anti-HER-2 antibody and a DR5 and HER-2 molecule, respectively, as described by the following assay. binding affinity in a DR5 or HER-2 Fab solution by balancing a Fab with a minimal concentration of labeled DR5 or HER-2 (I125), in the presence of a non-labeled serial titration of DR5 or HER-2, respectively, or by capturing molecules linked to DR5 or HER-2 respectively with an anti-Fab antibody coated plate (Chen, et al., (1999) J. Mol Biol 293: 865-881). To establish the conditions for the assay, microtiter plates (Dynex) are coated over night with 5 µg / ml anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked. with 2% (w / v) bovine albumin in PBS for two to five hours at room temperature (about 23 ° C). In an unabsorbed plate (Nunc # 269620), 100 pM or 26 pM DR5 or HER-2 [I125] is mixed with at serial dilutions of Fab of interest, for example Fab-12 (Presta et al., (1997) Cancer Res. 57: 4593-4599). The Fab of interest is then incubated overnight, however incubation may continue for 65 hours to ensure equilibrium is achieved. The mixtures are then transferred to the capture plate for incubation at room temperature for one hour. The solution is then withdrawn and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates are dry, 150uL / well of scintillant (MicroScint-20; Packard) is added, and the plates are counted in a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that are less than 20% of the maximum binding are chosen to be used in competition binding assays.

According to another embodiment Kd or Kd value can be measured by surface plasmon resonance assay using BIAcore ™ -2000 or a BIAcore ™ -3000 (BlAcore, Inc., Piscataway, NJ). In one example, the Kd value of the anti-DR5 or anti-HER-2 antibody molecule is determined using a BlAcore ™ analysis according to the following protocol. Briefly, carboxymethylated bistensor biosensor chips (CM5, BlAcore Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysucinimide (SNS) according to the supplier's instructions. Human DR5 or HER-2 is diluted with 10 mM sodium acetate, pH 4.8, to 5 µg / ml (~ 0.2 µM) before injecting at a flow rate of 5 µl / minute to approximately 10 units. coupled protein response (UK). Following injection of human DR5 or HER-2, 1M ethanolamine is added to block unreacted groups. For kinetic mediation, serial dilutions without duplicate Fab (0.78 nM and 500 nM) are injected into PBS with 0.05% Tween-20 (PBST) at 25 ° C at a flow rate of about 25 µl / min. . Association velocity (kon) and dissociation velocity (k0ff) are calculated using a Langmuir one-to-one single bond model (BlAcore Evaluation Software version 3.2) by simultaneously assembling association and dissociation desensograms. The equilibrium dissociation constant (Kd) is calculated as the k0ff / knn-Vide ratio, for example, Chen, Y., et al. (1999) J. Mol Biol 293: 865-881.

A "disorder" is any condition that could benefit from treatment with a substance / molecule or method of the invention. This included acute and chronic conditions or diseases including those pathological conditions that predispose the mammal to the disorder in question.

Nonlimiting examples of diseases to be treated herein include malignant and benign tumors; lymphoid and non-leukemic malignancies, neuronal, glial, astrocytic, hypothalamic, and other glandular, macrophage, epithelial, stromal, and blastocellic disorders; and inflammatory disorders and immune related diseases.

The terms "cell proliferation disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. As an example, the proliferative disorder is cancer.

"Tumor" as used herein refers to the multiplying growth of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferation disorder", "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

The term "cancer" and "cancerous" refers to or describes physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer. , ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, liver cancer, prostate cancer, vulval cancer, Thyroid cancer, liver carcinoma, and various types of head and neck cancer.

The term "related immune disease" means a disease where a component of a mammal's immune system causes, mediates or otherwise contributes to mammalian morbidity. Also included are diseases that stimulate or interfere with the immune response having an ameliorative effect on disease progression. Included with this term are autoimmune diseases, immune-mediated inflammatory diseases, infectious diseases, and immunodeficiencies. Examples of related diseases and inflammatory diseases, some of which are immune or T-cell mediated, which may be treated according to the invention include lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, sclerosis systemic (scleroderma), idiopathic inflammatory myopathy (dermatomyositis). , polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenia purpura, thyroiditis disease, thyroiditis, juvenile thyroiditis, juvenile thyroiditis) diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating disease of the central and peripheral nervous system, such as multiple sclerosis, idiopathic demyelinating disease or Guillain-Barré syndrome and polyneuropathy chronic inflammatory demyelinating, patellar diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, associated fibrotic and inflammatory responses, inflammatory bowel disease (Crohn's disease, ulcerative colitis), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases, including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis; allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria; immune diseases of the lung, such as eosinophilic pneumonia.

The term "autoimmune disease" is used broadly herein, in general the meaning refers to disorders or conditions in mammals in which destruction of healthy or normal tissues occurs from the mammalian cellular or humoral immune response against its own tissue. Examples include, but are not limited to, systemic lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, diabetesautoimmune, and inflammatory bowel disease (IBD).

As used herein, "treatment" indicates clinical intervention in an attempt to alter the natural course of the individual or the treated cell and may be performed for prophylaxis or during the course of clinical pathology. Desirable treatment effects include prevention of disease recurrence or recurrence, symptom relief, reduction of any direct or indirect disease pathological consequences, prevention of metastasis, reduction in disease progression, improvement or reduction of disease and remission, or better prognosis. In some embodiments, the antibodies of the invention are used to delay the development of disease or disorder.

"Effective amount" means an effective amount, at dosages and for periods of time necessary, to achieve the desired therapeutic or profilatic outcome.

A "therapeutically effective amount" of the substance or molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, gender and weight of the subject, as well as the ability of the antibody to permit desired reaction in the subject.

A therapeutically effective amount is also one in which any toxic or detrimental effect of the antibody is outweighed by the therapeutically beneficial effects. "Prophylactically effective amount" means the effective amount, in dosages and for periods of time required, to achieve the desired prophylactic result. Typically, as the prophylactic dose is used in patients before or in the initial stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

"Mammalian", for treatment purposes, means any animal classified as a mammal, including humans, farm and domestic animals, zoo, sporting or domestic animals, such as dogs, horses, cats, cows, etc.

The term "anti-neoplastic composition" refers to a composition useful in the treatment of cancer including at least one active therapeutic agent, for example, "anti-cancer agent." Examples of therapeutic agents (anti-cancer agents) include, but are not limited to, for example, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiotherapy agents, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents. for the treatment of cancer, such as anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonist (eg, a tyrosine kinase inhibitor), HER1 / EGFR inhibitors (eg, erlotinib (Tarceva ™), platelet-derived growth factor inhibitors (eg Gleevec ™ (Imatinib Mesylate)), a COX-2 inhibitor (eg celecoxib), interferons, cytokine antagonists (eg neutralizing antibodies) that bind to a or more of the following receptor targets ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BC MA or VEGF, TRAIL / Apo2 and other bioactive and biological chemical agents and etc. Combinations are also ineffective. included in the invention.

The term "labeled epitope" as used herein refers to a mutant antibody fused to an "epitope tag". The epitope-marking polypeptide has sufficient residues to provide an epitope against which an antibody against it can be made, yet is short enough that it does not interfere with the activity of the mutant antibody. The epitope marker preferably is also quite unique so that the antibody does not cross react against other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and generally between about 8-50 amino acid residues (in certain embodiments, between about 9-30 residues). Examples include, but are not limited to, flu HA marker polypeptide and its 12CA5 antibodies (Field et al. Mol. Cell. Biol. 8: 2159-2165 (1988)); the c-myc marker and antibodies thereto 8F9, 3C7, 6E10, G4, B7 and 9E10 (Evan et al., Mol. Cell. Biol. 5 (12): 3610-3616 (1985)); and the Herpesvirus glycoprotein D (gD) marker and its antibodies (Paborsky et al, Protein Engineering3 (6): 547-553 (1990)). In certain embodiments, the deepitope tag is a "recovered receptor-linked epitope".

The term "cytotoxic agent" as used herein denotes a substance that inhibits or prevents cell function and / or causes cell destruction. The term is intended to include radioactive isotopes (such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 Lu radioactive isotopes), chemotherapeutic agents, for example methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine , etoposide), doxorubicin, melfalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof, such as ezimasnucleotides, antibiotics and toxins such as small detoxin molecules or ezmatically active toxins of bacterial, fungal origin plant or animal, including fragments or variants thereof, and various anti-cancer or anti-tumor agents described below. Other cytotoxic agents are described below. A massive tumoricide causes destruction of the tumor cells.

A "chemotherapeutic agent" is a useful chemical compound for treating conditions such as cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan®), alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and ethylamellamines, including altretamine, triethylenomelamine, triethylenophosphoramide, triethylenephosphoramide and trimethylolomelamine, acetogenins (especially bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacone; lapacol; colcicines; betulinic acid; camptothecin (including the synthetic analog topopotecan (HYCAMTIN®)), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9- (aminocamptothecin); bryostatin; calistatin; CC-1065 (including their adozelesin, carzelesin and byzelesine synthetic analogues); podophyllotoxin; podophilic acid; teniposide (cryptophycin particularly cryptophycin 1 and cryptophycin 8); dolastatin, duocarmycin (including synthetic analog, KW-2189 and CB1-TM1); pancratistatin; sarcodictiin; spongistatin; denitrogen mustards, such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melfalan, novembiquin, phenesterine, prednimustine, trophosfamide, uromile mustard, such as, carmustine, photomustine ranimustine; antibiotics, such as enedin antibiotics (eg caliqueamicin, especially caliqueamicin gamai I and caliquemicinomegall; see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); dinemicin, including dinemicin A speramycin; as well as neocarzinostatinachromophore and chromoprotein-related antibiotic enedin chromophores), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, chromomycin, 5-dunorubicin, detoxinin -norleukin, cyanomorpholine-doxorubicin, 2-pyrroline-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as, for example mitomycin C, mycophenolic acid, nogalamycin, stromalcinine, mycobacterine, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, and 5-fluorouracil (5 -FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; glycoside aldophosphamide; aminolevulinic acid, amsacrine; bestrabucil; bisantrene; edatraxate; defopamine; demecolcin diaziquone; elfornitine; elliptin acetate; epothilone; etoglucide; degassium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and eanamytocins; mitoguazone; mitoxantrone; mopidamol; nitacrine; pentostatin fenamet; pirarubicin; podophilic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2 ', 2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; manomustine; mitobronitol; mitolactol; pipobromanine; gacytosine ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM albumin-constructed paclitaxel nanoparticles formulation (AmericanPharmaceutical Partners, Schaumberg, Illinois), and doxetaxel TAXOTERE® (Rhône-Poulenc Rorer, Antony, France); chlorambucil (GEMZAR®); gemcitabine; 6-thioguanine (VELBAN®); mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornitine; apecitabine (XELODA®); and pharmaceutically acceptable bones, acids or derivatives of any of the above; as well as combinations of two or more of the agents described above such as, for example CHOP, an abbreviation for the combined therapy of decyclophosphamides, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for 5-FU combined oxaliplatin treatment regimen (ELOXATINTM) and leukovovine.

Also included in this definition are anti-hormonal agents that act to regulate, reduce, obstruct, or inhibit the effects of hormones that can promote cancer growth, and are often systemic or whole-body treatment. These may be the hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX otamoxifene), raloxifene (EVISTA®), droloxifene, 4-hydroxyitamoxyphene, trioxifene, keoxifene, LY117018, LY117018 , onapristone, and toremifene (FARESTON®); anti-progesterones; estrogen receptor suppressor regulators (ERDs); agents that function to suppress the ovaries, for example, luteinizing hormone-releasing agonist hormone (LHRH) such as LUPRON® and ELIGARD® leuprolide acetates, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens such as aflutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands such as 4 (5) -imidazoles, aminoglutethimide, acetate demegestrol (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RIVISOR ®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®). In addition, such definition of chemotherapeutic agents includes bisphosphonates such as oclodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®) orisedronate (ACTONEL®); as well as troxacytabine (a cytosine analogous 1,3-dioxolane analog); antisense oligonucleotides, particularly those that inhibit expression of the signaling pathway genes, implying aberrant cell proliferation such as PKC-alpha, Raf, H-Ras and the epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; datopoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (an ErbB-2 and a small kinase inhibitor EGFR molecule also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

"Growth inhibitory agent" as used herein means the compound or composition that inhibits cell growth, especially of the cancer cell expressing the target molecule in vitro or in vitro. Thus, the growth inhibitory agent can significantly reduce the percentage of cells expressing the target molecule in phase S. Examples of growth inhibitory agents include agents that block cell cycle progress (at a different site than the S phase), such as agents that induce the G1 suspension and M phase suspension. Class M phase blockers include creases (vincristine and vinblastine), taxanes and top II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. G1 suspending agents also diffuse into S-phase suspension, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Additional information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled Cell Cycle Regulation, Oncogenes and Antineoplastic Drugs by Murakami et al (WB Saunders: Philadelphia, 1995), especially p.13. Taxanes (paclitaxel and docetaxel) are the two anti-neoplastic drugs derived from the yew tree. Docetaxel (TAXOTERE ®, Rhone-Poulenc Rorer), obtained from European yew, is a semi-synthetic paclitaxel analog (Taxol ®, Bristol-Myers Squibb). Docetaxel and paclitaxel promote microtubule assembly of tubulin dimers and stabilize microtubules, preventing depolymerization, which results in inhibition of mitosis in cells.

"Doxorubicin" is an anthracycline antibiotic. The full name of the chemical product is doxorubicin (8S-cis) -10 - [(3-amino-2,3,6-trideoxy-aL-trash-hexapyranosyl) oxy] -7,8,9,10-tetrahydro-6, 8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphtacenedione.

The term "prodrug" as used herein refers to a precursor or derivative form of a reactive pharmaceutical substance that is less cytotoxic to tumor cells compared to parent drug and is capable of being enzymatically activated or converted to the most active parent form. See, for example, Wilman, Prodrugs in Cancer Chemotherapy, Biochemical Society Transactions, 14, p. 375-382, 615a Belfast Meeting (1986) and Stella et al., Prodrugs: A Chemical Approach to Drug Delivery, Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Human Press (1985). Prodrugs according to the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-modified prodrugs. -amino acids, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into the free drug cytotoxic drug. Examples of cytotoxic drugs that may be derivatized in drug form for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

For the treatment of rheumatoid arthritis ("RA"), the patient may be treated with an antibody of the invention in conjunction with any of the following drugs: DMARDS (disease modifying antirheumatic drugs (such as methotrexate)), NSAI or NSAID ( non-steroidal anti-inflammatory drugs), Humira ™ (adalimumab; Abbott Laboratories), Arava® (leflunomide), Remicade® (infliximab; Centocor Inc. of Malvern PA, United States), Enbrel (etanercept; Immunex, WA, United States), COX inhibitors -2. DMARDs commonly used in RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine and staphylococcus protein A. Adalimumab is a human antibodies to TNF-γ binding. Infliximab is a monoclonal antibody that binds to TNF. Etanercept is an "immunoadhesin" fusion protein consisting of the extracellular binding portion of the 75 kD human tumor necrosis factor (p75) tumor receptor (TNFR) ligand bound to the Fc portion of a human IgG1. For conventional treatment of RA, see, for example, Guidelines for the Management of Rheumatoid Arthritis, Arthritis & Rheumatism 46 (2): 328-346 (February 2002). In a specific embodiment, the RA patient is treated with an anti-CD20 antibody according to the present invention together with methotrexate (MTX). An example MTX dosage is about 7.5 to 25 mg / kg / week. MTX may be administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, the patient may be treated with an antibody of the invention together, for example with Remicade® (infliximab; from Centocor Inc. of Malvern PA, United States), and / or Enbrel (etanercept; Immunex WA, United States).

For the treatment of SLE, the patient may be treated with an antibody of the invention in conjunction with, for example, a high dose of corticosteroids and / or cyclophosphamide (HDCC).

For the treatment of psoriasis, patients may receive an antibody of the present invention in conjunction with topical treatments such as topical steroids, anthraline, calcipotriene, clobetasol and tazarotene, or with methotrexate, retinoids, cyclosporine, PUVA and UVB therapies. In one embodiment, the psoriasis patient is treated with the antibody of the invention either sequentially or simultaneously with cyclosporine.

"Isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid encoding the antibody. Isolated nucleic acid molecule is different from the form in which it is found in nature. Isolated nucleic acid molecules are therefore differentiated from the existing nucleic acid molecule in natural cells. However, isolated nucleic acid molecule includes nucleic acid molecule contained in cells that normally express the antibody in which, for example, the "isolated" nucleic acid molecule is at a different chromosomal site than natural cells.

The term "control sequences" refers to DNA sequences required for expression of the operably linked coding sequence in a specific host organism. Control sequences that are suitable for prokaryotes, for example, include promoter, optionally operator sequence, and deribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

The "variant" or "mutant" of an initial or reference polypeptide (for example, a source antibody or its variable domain (s) / CDR (s)), such as a fusion protein (polypeptide) or a heterologous polypeptide (phage heterologous), is a polypeptide which: 1) has an amino acid sequence different from the initial or reference polypeptide and 2) was derived from an initial or reference polypeptide, either by the mutation of natural or artificial (anthropic) polypeptide ). Such variants include, for example, deletions of, and / or insertions into, and / or substitution of residues in the amino acid sequence of the polypeptide of interest. For example, a fusion polypeptide of the invention produced using an oligonucleotide comprising a restricted codon set encoding a sequence with a variant amino acid (with respect to the amino acids found at the corresponding positions of an antisense antibody / fragment fragment) would be a related variant polypeptide. to a source antibody and / or antigen binding fragment and / or a CDR. Thus, a variant CDR refers to a CDR that has a variant sequence with respect to the initial or reference polypeptide sequence (such as an antigen binding and / or CDR binding antibody / fragment). An amino acid variant in this context refers to an amino acid other than amino acid at the corresponding position in an initial or reference polypeptide sequence (such as that of an antigen-binding and / or CDR binding antibody / fragment). Any combination of deletions, insertions, substitutions may be performed to arrive at the construction of the final mutant variant, provided that it has the desired functional characteristics. In some of the examples described herein, ligand sequences have point mutations as deletions or additions. Modifications of amino acid can also occur by post-translational polypeptide processes, such as a change in the number or position of the glycosylation sites. Methods for producing variant polypeptide amino acid sequences are described in US Patent 5,534,615, expressly incorporated herein by reference.

A "wild type" sequence or "reference" sequence or sequence of a "wild type" polypeptide / protein or sequence of a "reference" polypeptide / protein, such as a capsid protein, or a CDR variable domain of a source antibody, may be the reference sequence from which the variant polypeptide sequence is derived by the introduction of mutations. In general, the "wild type" sequence for a given protein sequence is the sequence that is most common in nature. Likewise, a "wild type" genetic sequence is the degene sequence that is most commonly found in nature. Mutations can be introduced into a "wild-type" gene (and therefore encoding asproteins), both by natural and man-induced processes. The products of such processes are "variant" or "mutant" forms of the original "wild-type" gene or protein.

A "variety" of a substance, such as a polypeptide or polynucleotide of the invention as used herein, generally refers to a collection of two or more types or forms of the substance. There are two or more types or forms of a substance if two or more of the substances seduce each other with respect to a particular characteristic, such as a variant amino acid found at a given amino acid position. For example, a variety of polypeptides of the invention exist, if there are two or more polypeptides of the invention that are substantially the same, or are identical in sequence except for the sequence of a CDR variant or except for an amino acid variant in a highly diverse and accessible amino acid position. to the solvent. In another example, there is a variety of polynucleotides of the invention, if there are two or more polynucleotides of the invention that are basically the same or identical in sequence except for the sequence encoding a variant CDR or except for amino acid variant at a highly diverse and solvent-accessible amino acid position. .

The invention provides a novel method for producing and isolating polypeptides that bind to a target antigen, such as antibodies or antigen-binding fragments, which may have a high affinity for a selected antigen. A variety of different linker polypeptides are prepared by mutation (diversification) at one or more selected amino acid positions in a light chain and / or heavy domain variable source antibody with a restricted codon set to generate a library of variant amino acid linker polypeptides at at least least one CDR sequence in which the number of variant amino acid types is kept to a minimum (ie 19 or less, 15 or less, 10 or less, 8 or less, 6 or less, 4 or less, or 2, but in general at least 2). Amino acid exposures include those that are accessible to the solvent, for example, as determined by structural analysis of a highly diverse source and / or ques- tion antibody and / or naturally occurring immunoglobulin polypeptides. Another advantage offered by the limited nature of the diversification of the invention is that additional amino acid positions other than those highly diversified and / or solvent accessible may also be diversified according to the need or desire of the practitioner; Examples of these embodiments are described herein.

The highly diverse solvent-accessible amino acid positions in certain embodiments are those in the CDR region variable domains of the antibody selected from the group consisting of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof. Amino acid positions are each mutant using a restricted decon set that encodes a limited number of amino acids, the choice of amino acids is generally independent of the amino acids commonly occurring at each position. In some exemplary embodiments, when a highly accessible or highly accessible solvent arrangement in a CDR region is mutated, a codon set is selected to encode from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, and / or only 2 amino acids. In some embodiments, when a solvent accessible or highly diversified position in a CDR region is mutated, a codon set is selected to encode from 2 to 19, 2 to 15, 2 to 10, 3 to 9, 4 to 8, and / or from 5 to 7 amino acids. In some embodiments, a conjunction codon encodes at least 2, but 19 or less, 15 or less, 10 or less, 8 or less, 6 or less, 4 or fewer amino acids. CDR sequences can also be diverse, varying in length. For example, for CDRH3, CDRH3 variant regions that can be generated are of different lengths and / or are randomized at the selected positions using a restricted codon set.

Library diversity of CDR variant-containing polypeptides is constructed using a codon set that encodes only a limited number of amino acids, for example, a minimal but sufficient amount of sequence variety is introduced into a CDR. The number of mutant positions in the CDR is minimized and the variant amino acids at each position are designed to include a limited number of amino acids, regardless of amino acids considered as commonly occurring at known or naturally occurring positions in the CDRs. In a specific embodiment, a single antibody, including at least one CDR, is used as the source antibody. It is surprising that an antibody variable domain library having sequence and size diversity can generate using a single antibody font as a template and reaching a variety of specific positions using a limited number of unconventional amino acid substitutions.

Development of Antibody Variable Domains Diversity

In one aspect of the invention, high quality libraries of antibody variable domains are generated. Libraries have restricted sequence diversity of different CDR sequences, for example, the diversity of antibody variable domains. Libraries include variable domains of high affinity binding antibodies for one or more antigens, including, for example, human DR5 and HER-2. Library diversity is designed by selecting amino acid positions that are solvent-accessible and highly diverse in a single source antibody and mutating its positions in at least one CDR using a restricted codon set. The restricted codon set may, in certain embodiments, encode less than 19, 15, 10, 8, 6, 4 amino acids or encode only 2 amino acids. One source antibody is humanized 4D5 antibody, but the methods for diversification may be applied to other source antibodies whose frequency is known. A source antibody may be a naturally occurring antibody, synthetic antibody, recombinant antibodies, humanized antibody, germline derived antibody, chimeric antibody, affinity matured antibody or antigen binding fragment. Antibodies can be obtained from a variety of mammalian species, including humans, mice and rats. In some embodiments, a source antibody is an antibody that is obtained after one or more initial affinity selection steps, but prior to one or more affinity maturation step (s). A source antibody may be selected or modified to be provided for high productivity and stability when produced in cell culture.

The 4D5 antibody is a humanized cancer-specific antigen specific antibody known as HER-2 (erbB2). The antibody includes variable domains having the consensus structural region; few positions were reversed to the mouse sequence during the process to increase the affinity of the humanized antibody. The sequence and crystal structure of the humanized 4D5 antibody are described in US Patent 6,054,297, Carter et al, PNAS 89: 4285 (1992), the crystal structure is shown in J Mol. Biol. 229: 969 (1993) and online at http: //www.ncbi. nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&dB=t&Dopt=s&uid=990,http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&dB= t & Dopt = s & uid = 991, ehttp: //www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi? form = 6 & db = t & Dopt = s & uid = 992.

One criterion for producing a diversity in antibody variable domains is mutating residues at positions that are accessible to the solvent (as defined above). These positions are usually found in CDRs, and are typically found outside proteins. In certain embodiments, solvent accessible positions are determined using coordinates in 3-dimensional antibody models using a computer program such as Insightll (Accelrys, San Diego, California). Solvent-accessible positions can also be determined using state-of-the-art algorithms (e.g., and Lee Richards, J. Mol. Biol. 55, 379 (1971) and Connolly, J.Appl. Cryst. 16, 548 (1983)) . Determination of solvent accessible positions can be performed using software suitable for modeling and obtaining the three-dimensional structural information of the protein from an antibody. Software that may be used for these purposes includes SYBYLBiopolymer Module software (Tripos Associates). Generally, in certain embodiments, where an algorithm (program) requires the use of an input size parameter, the "size" of a probe that is used in the calculation is fixed at about 1.4 Angstrom or less. In addition, the determination of solvent-accessible regions using and area methods using personal computer software has been described by Pacios ((1994) "ARVOMOUCONTOUR: molecular surface areas and volumes on PersonalComputers." Comput. Chem. 18 (4): 377- 386; and (1995). "Variations of Surface Areas and Volumes in Distinct Molecular Surfaces of Biomolecules." J. Mol.Model. 1: 46-53.)

In some cases, the selection of solvent accessible residues is further refined by the choice of solvent accessible residues which collectively form a minimal contiguous region, for example when the reference polypeptide or source antibody is in its 3-fold structure. For example, as shown in Figure 2, a compact (minimal) contiguous region is made up of residues selected for humanized 4D5 CDRH1 / H2 / H3 / L1 / L2 / L3. A (minimal) contiguous contiguous region set may include only a subset (e.g., CDRs 2-5) of the full range of CDRs, for example, CDRH1 / H2 / H3 / L3. Solvent-accessible residues that do not contribute to the formation of such a region may optionally be excluded from diversification. Refinement of the selection by this criterion allows the trader to minimize, as desired, the number of wastes to be diversified. For example, residue 28 on H1 may optionally be excluded in diversification since it is at the end of the region. However, this criterion may also be used, whenever desired, to choose residues to be diversified which may not necessarily be considered accessible to the solvent.

For example, a residue that is not considered accessible to the solvent but forms a contiguous region in the 3-D structure that is coiled with other residues that are considered accessible to the solvent may be selected for diversification. An example of this is CDRL1-29. Selection of such residues will be apparent to one skilled in the art, and their suitability can also be determined empirically and according to the needs and wishes of the skilled technician.

The solvent accessible positions identified from the crystal structure of the humanized 4D5 antibody for each CDR are as follows (residue position according to Kabat):

CDRL1: 28, 30, 31.32

CDRL2: 50.53

CDRL3: 91, 92, 93, 94, 96

CDRH1: 28, 30, 31.32, 33

CDRH2: 50, 52, 52, 53, 54, 55, 56, 57, 58.

In addition, in some embodiments, CDRL1 residue 29 may also be selected based on their insertion into a contiguous region that includes another solvent accessible residue. All or a subset of positions accessible to the solvent set forth above may be further diversified according to the methods and compositions of the invention. For example, in some embodiments, only positions 50, 52, 52,53-56, and 58 are randomized to CDRH2.

Another criterion for selecting positions to be mutated are positions that reveal variability in the amino acid sequence when purchased with the sequences of a known and / or natural antibody. A highly diverse position refers to an amino acid position located in the light or heavy chain variable region that has a number of amino acids different from that represented at the amino acid position of amino acid sequences when compared to the known and / or natural ligand antibody or fragment. Highly diversified positions may be in the CDR region. CDRH3 positions are all considered highly diversified. In certain embodiments, amino acid residues are highly diversified if they are from about 2 to 19 (although numbers may vary as described herein) possible different variations of amino acid residues at such a position.

In one aspect, the identification of highly diversified positions in known and / or naturally occurring antibodies is facilitated by data provided by Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md, 1987 and 1991). An Internet-based database located at http://www.bioinf.orq.uk.abs.structures.html offers an extensive collection and alignment of light chain sequences (http://www.biomf.orq.uk.abs). lc.align) and heavy chain (http //: www.bioinf.orq.uk.abs.hc.aliqn) and facilitates the determination of highly diversified positions in these sequences. The diversity in the solvent-accessible positions of the humanized 4D5 antibody in known and / or naturally occurring heavy and light chains is shown in Figures 3 and 4.

In one aspect of the invention, highly diverse and solvent-accessible residues in at least one, two, three, four, five or all CDRs selected from the group consisting of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof are mutant. (ie, randomized using a restricted codon set as defined herein). For example, a population of polypeptides may be generated by diversifying at least one solvent accessible and / or highly diversified residue into CDRL3 and CDRH3 using a restricted codon set. Thus, the invention provides a large number of novel substitution-formed antibody sequences from at least one highly diverse solvent accessible position of at least one source antibody variable domain CDR with amino acids encoded by a restricted codon. For example, a CDR variant or antibody variable domain may represent an amino acid variant at one or more positions of amino acids 28, 30, 31, 32, 33, and / or 34 of CDRH1; and / or one or more of amino acids 50, 52, 52, 53, 54, 55, 56 and / or 58 of CDRH2; and / or at one or more amino acid positions 95-100, 100a, 100b, and 100c of CDRH3; and / or at one or more positions of amino acids 28, 29, 30 and / or 31 of CDRL1; and / or at one or more positions of amino acids 50 and / or 53 in noCDRL2; and / or at one or more amino acid positions 91, 92, 93, 94, 95e and / or 96 in CDRL3. In another example, a CDR variant or antibody variable domain may consist of an amino acid variant at one or more positions of amino acids 28, 30, 31, 32, and / or 33 of CDRH1; and / or at one or more positions of amino acids 50, 52, 53, 54, 56 and / or 58 of CDRH2, and / or at one or more amino acid positions 95-100, 100a, 100b and 100c of CDRH3; and / or at one or more positions of amino acids 28, 29, 30 and / or 31 of CDRL1; and / or at one or more positions of amino acids 50 and / or 53 in noCDRL2; and / or at one or more amino acid positions 91, 92, 93, 94 and / or 96 in CDRL3. Variant amino acids at these positions are encoded by the restricted codon set as described herein.

As discussed above, amino acid variants are encoded by a restricted codon set. A codon set is a set of different nucleotide triad sequences used to encode the desired variant amino acid group. Synthesis of oligonucleotides with the "degeneracy" of selected nucleotides at certain positions is well known in the art. Such oligonucleotide sets with certain codon sets may be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, California), or may be commercially obtained (for example, from Life Technologies, Rockville, MD).

Therefore, a set of synthesized oligonucleotides having a specific decon set will normally include a variety of oligonucleotides with different sequences, the differences established by the decon set within the overall sequence. Oligonucleotides, as used in the present invention, have sequences that allow hybridization of a template to a variable domain nucleic acid and may also, but not necessarily, include sites useful for the use of restriction enzymes, for example for cloning purposes.

In one aspect, the restricted repertoire of amino acids intended to occupy one or more of the highly accessible solvent positions in CDRs of the humanized 4D5 antibody are determined (based on the wishes of the practitioner, which may be based on a number of criteria, including specific amino acids). for desired determinations, absence of specific amino acid (s) in desired determinations, desired library size, characteristic of antigen binding binders, etc.)

Heavy chain CDR3s (CDRH3s) in known antibodies have several sequences, structural conformations, and lengths. CDRH3s are often found in the middle of the antigen binding area and often participate in contact with the antigen. The elaboration of CDRH3 can thus be developed separately from that of other CDRs, as it can be difficult to predict the structural conformation of CDRH3 and the amino acid diversity in this region is especially recognized in several antibodies. In accordance with the present invention, the CDRH3 is designed to generate diversity at specific positions within CDRH3, for example at positions 95, 96, 97, 98, 99100, 100a, 100b, and 100c (e.g. according to Kabat numbering). on antibody 4D5).

In some embodiments, diversity is also generated by the lengthening of CDRH3 using a restricted codon set. The length diversity may be of any empirically determined range as it is suitable for generating a population of polypeptides that contain large proportions of antigen binding proteins. For example, polypeptides comprising the CDRH3 variant can be generated with the sequence X1-X2-X3-X4-X5- (X6) n-X7-X8-X9-DY (SEQ IS No: 4), in which X1-X9 are amino acids encoded by the restricted codon set, n and are of different lengths, for example, n = 1-11, 5-11 or 7-11. Other examples of possible n-values are: 1,2, 3, 4, 5, 6 , 7, 8, 9, 10 and 11.

Illustrative embodiments of oligonucleotides which may be used to provide a variety in length of the CDRH3 sequence include those indicated in Figure 9A-9D, Figure 20A-L and Figure 23.

It is contemplated that library sequence diversity created by introducing amino acid variants into a specific CDR, for example, CDRH3, can be increased by combining the CDR variant with other CDRs that have variations from other antibody regions, specifically other CDRs. of both light and heavy chain variable sequences. It is also contemplated that nucleic acid sequences encoding members of this set may be further diversified by introducing other variant amino acids into CDRs, both light and heavy chain variable sequences, by codon assemblies. Thus, in one example, CDRH3 sequences of fusion polypeptides that bind to the target antigen may be combined with diverse CDRL3, CDRH1, or CDRH2 sequences, or any combination of diverse CDRs.

It should be noted that in some cases the residues of the structural region may be varied with respect to the sequence of an antibody source or antigen-binding fragment, for example to reflect consensus sequence or to improve stability or display. For example, heavy chain residues in the structural region 49, 93, 94, or 71 may serve. The heavy chain residue 93 of the framework region may be seralanine or serine (which is the human consensus amino acid sequence at this position.). The heavy chain residue 94 of the structural region may be altered to reflect the consensus sequence of the structural region from threonine to arginine or lysine. Another example of structural region residues that can be altered is structural region heavy chain residue 71, which is R in about 1970 polypeptides, V in about 627 polypeptides, and A in about 527 polypeptides, as found in the database. data from Kabat. The heavy chain residue 49 of the framework region may be oralanine glycine. In addition, optionally, the 3 N-terminal amino acids of the heavy chain variable domain may be removed. In the light chain, optionally, the amino acid arginine at position 66 may be modified to paraglycine. In one example, residue 93 in the structural region heavy chain is alanine and residue 94 is arginine.

to produce fusion polypeptides that bind with significant affinity to potential ligands. These constructs are dimerizable domains that when present in a fusion polypeptide provide an increased tendency to dimerize heavy chains to form Fab or Fab 'antibody defragmenting dimers or portions. These dimerization domains may include, for example, a heavy chain hinge sequence (for example, a sequence that includes TCPPCPAPELLG (SEQID No: 5) which may be present in the fusion polypeptide). The dimerization domains in the phage fusion polypeptides bring two sets of joined fusion polypeptides (LC / HC-phage protein / fragment (such as polyl1)), thus allowing the formation of appropriate bonds (such as heavy chain disulfide bridges) between the two. fusion polypeptide sets. The constructed vector containing such dimerization domain may be used to achieve divalent exposure of the antibody variable domain, for example, the various fusion proteins described herein, in a phage. In certain embodiments, the intrinsic affinity of each monomeric antibody fragment (fusion polypeptide) is not significantly altered by fusion to the dimerization domain. In certain embodiments, the dimerization results in the display of a phage equivalent, which provides an avidity increase in phage binding, with a significant decrease in dissociation rate, which can be determined by methods known in the art and as described herein. Vectors containing the dimerization domain of the invention may or may not also include an amber stop codon after the dimerization domain. Dimerization may be varied to achieve different characteristics. Dimerization domains may include a cysteine residue-containing sequence, a full-length anti-antibody hinge region, such as a dimerization sequence such as leucine or GCN4 zipper sequence or mixtures thereof. Dimerization sequences are known in the art, and include, for example, the zipper sequenceGCN4 (GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG) (SEQ ID No: 3). The dimerization domain in certain embodiments is located at the C-terminal end of the variable domain sequence or heavy chain constant and / or between the variable domain sequence or heavy chain constant and any sequence of the viral dacapsid protein component. An amber stop codon may also be present at the end of the C-terminal end of the dimerization domain. In one embodiment, where the amber stop codon is present, the dimerization domain encodes at least one cysteine and a dimerization sequence such as a leucine zipper. In other embodiments, where the amber-faced codon is not present, the dimerization domain may include a single cysteine residue.

The polypeptides of the invention may also be fused to other types of polypeptides to suit them for displaying polypeptide variants or to provide for polypeptide purification, selection or screening and detection. For an example involving phage display, the polypeptides of the invention are fused to all or part of a viral capsid protein. Examples of viral capsid proteins include the p111 proteins, pVIII main protein, Soc, Hoc, gpD, pv1, and variants thereof. In addition, variant polypeptides produced according to the methods of the invention may optionally be fused to a labeling or labeling polypeptide. such as FLAG, polyhistidine, gD, c-myc, B-galactosidase and the like.

Methods for Producing Randomized Variable Domain Libraries

Methods for substituting an amino acid of choice in a model nucleic acid are well established in the state of the art, some of which are described herein. For example, libraries may be designed to achieve solvent-accessible and / or highly diverse positions at least one amino acid substitution in the CDR variant region using the Kunkel method. See, for example, Kunkel et al., MethodsEnzymol. (1987), 154: 367-382. The production of randomized sequences is also described below in the Examples.

The oligonucleotide sequence includes one or more of the restricted codon sets designed for different lengths of CDRH3 or for solvent-accessible or highly diverse positions in a CDR.

A codon set is a set of different denucleotide triad sequences used to encode the desired amino acid variant. A codon set can be represented using symbols to designate a specific nucleotide or equimolar nucleotide mixtures as shown below according to the IUB code. . Typically, a codon set is represented by three uppercase letters, for example, KMT, TMT, and the like.

IUB code

Guanine A adenineT thymineC CytosineR (A or G) Y (C or T) M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T)

N (A or C or G or T)

For example, in the TMT codon set, T is nucleotide thymine; M may be A or C. This codon set may have multiple codons and may encode only a limited number of amino acids, ie serine and tyrosine.

An oligonucleotide set or primer can be synthesized using standard methods. An oligonucleotide set may be synthesized, for example, by solid phase synthesis, containing sequences representing all possible combinations of nucleotide triads provided by the restricted codon set and encoding the desired amino acid restriction group. The synthesis of oligonucleotides with the "degeneracy" of selected nucleotides at certain positions is well known in the prior art. Such oligonucleotide sets with certain codon sets may be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, California), or may be obtained commercially (for example, from Life Technologies, Rockville, MD). Therefore, a set of synthesized oligonucleotides having a specific codon set will normally include a variety of different sequence oligonucleotides, the differences established by the codon set within the overall sequence. Oligonucleotides, as used in the present invention, have sequences that allow hybridization to a CDR (for example, those contained in a variable domain) of a nucleic acid template and may also include sites useful for the use of restriction enzymes, for example. , for cloning purposes.

In one method, nucleic acid sequences encoding amino acid variants can be created by polyolucleotide-mediated mutagenesis of a nucleic acid sequence encoding a source polypeptide or template such as the antibody variable domains4D5. This technique is well known in the state of the art as described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 (1987). Briefly, the coding of the variant amino acid nucleic acid sequences is created by hybridizing a set of oligonucleotides encoding the desired restricted codon set to a template DNA, wherein the template is a single-stranded plasmid form containing a template region of nucleic acid sequences. variable. After hybridization, the DNA polymerase is used to synthesize an entire second complementary template strand that will thus integrate the oligonucleotide primer, and will contain the restricted codon set, as provided by the oligonucleotide set. Nucleic acids that encode other source or template molecules are known or can be readily determined.

Generally, oligonucleotides of at least 25 long-acting nucleotides are used. An optimal oligonucleotide will have at least 12 to 15 nucleotides that are fully complementary to the template of both sides of the nucleotide (s) encoding the mutation (s). This ensures that the oligonucleotide will properly hybridize to the single-stranded template DNA molecule. Oligonucleotides are readily synthesized by techniques known in the art, as described by Crea et al., Proc. A / there / .Acad. Know. USA, 75: 5765 (1978).

The DNA template (Template DNA) is generated by vectors that are derived from bacteriophage M13 vectors (commercially available vectors M13mp18 and M13mp19 are suitable), or vectors that contain a single origin of single stranded phage replication, as described by Viera et al. , Meth. Enzymol., 153: 3 (1987). Thus, the DNA that will be mutated can be inserted into one of these vectors in order to generate a simple (template) template. The production of single ribbon mold is described in sections 4.21-4.41 of Sambrook et al., Above.

To alter the sequence of native DNA, the oligonucleotide is hybridized by a single stranded template under the appropriate hybridization conditions.

The DNA polymerization enzyme, usually DNA polymerase or Klenow fragment of DNA polymerase I, is then added to synthesize the complementary template cast using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one DNA strand encodes the mutant form of gene 1, and the other strand (the original model) encodes the unchanged native gene 1 sequences. This heteroduplex molecule is then used to modify a suitable host cell, usually a prokaryote such as E. coli JM101. After cell growth, they are seeded on selected agarose plates using a Phosphate-32 radiolabeled oligonucleotide primer to identify bacterial colonies that contain the mutated DNA.

The method described immediately above can be modified in detail so that a homoduplex molecule is created where both strands of the plasmid have mutation (s). Modifications are as follows: The simplex strand oligonucleotide is annealed to the single strand of the template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribothimidine (dTT), is combined with a thiodeoxyribocytosine modification called TCP- (aS) (obtainable from Amersham). This mixture is added to the template oligonucleotide complex. After the addition of the DNA polymerase in the mix, a DNA strand identical to the template strand except for the mutant bases is then produced. In addition, this new DNA strand will contain dCTP- (aS) instead of dCTP, which will protect it from restriction-endonuclease digestion. After the double-stranded heteroduplex template tape is cut with an appropriate restriction enzyme, the template tape can be digested with nucleaseExolll or other suitable nuclease, passing the region containing the site (s) to be mutagenized. (s). The reaction is then stopped to leave a molecule that is only partially single stranded. A complete double stranded heteroduplicate DNA is then formed using DNA polymerase, in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This heteroduplicate molecule can then be used to modify an appropriate host cell.

As indicated above the sequence of the oligonucleotide set is of sufficient length to hybridize nucleic acid and may, but may not necessarily include restriction sites.

The DNA template can be generated by vectors that are derived from M13 bacteriophage vectors or vectors that contain a single single stranded phage replication source, as described by Viera et al, Meth. Enzymol., 153: 3 (1987). Thus, the DNA that will be mutated can be inserted into one of these vectors in order to generate a simple ribbon template. The production of single tape mold is described in sections 4.21-4.41 of Sambrook et al., Supra.

According to another method, a library may be generated by providing sets of upstream and downstream oligonucleotides, each set with a variety of differently differing oligonucleotides, the different sequences established by the set of codons provided within the oligonucleotide sequence. The upstream and downstream oligonucleotide set, together with a variable domain template nucleic acid sequence, can be used in polymerase chain narration to generate a "library" of pCR products. PCR products may be referred to as "nucleic acid cassettes", and they may be fused to other related or unrelated nucleic acid sequences, for example, viral capsid protein component components, dimerization domains, using established molecular biology techniques.

The sequence of the PCR primers includes one or more of the codons designed for solvent accessible positions or highly diverse in a CDR region. As described above, a codon set is a set of different nucleotide triad sequences used to encode the desired variant amino acids.

The set of oligonucleotides may be used in a polymerase chain reaction using a variable region nucleic acid sequence for the creation of nucleic acid cassettes. Sequence of variable region template nucleic acids can be any portion of the immunoglobulin light or heavy chain variable region template nucleic acid sequence (ie, nucleic acid sequences encoding target amino acids for substitution). The variable region template nucleic acid sequence is a portion of a double-stranded DNA molecule having the first nucleic acid strand and the second complementary nucleic acid strand. The template nucleic acid sequence of the variable region contains at least a portion of a variable domain and has at least one CDR. In some cases, template nucleic acid sequence of the variable region contains more than one CDR. An upstream and downstream portion of a variable region template nucleic acid sequence may be targeted for hybridization with members of an upstream oligonucleotide set and a downstream oligonucleotide set.

A first oligonucleotide from a downstream primer assembly can hybridize the first nucleic acid strand and a second oligonucleotide from an upstream primer assembly can hybridize the second nucleic acid strand. Oligonucleotide primers may include one or more codon assemblies designed to hybridize a portion of the template variable region nucleic acid sequence. Use of these oligonucleotides may introduce two or more codon sets into the PCR product (ie nucleic acid cassettes). The oligonucleotide primer that hybridizes to the template variable region nucleic acid sequence encoding the antibody variable domain portions includes portions encoding CDR residues that are targeted for amino acid substitution.

Upstream and downstream oligonucleotide assemblies may also be synthesized to include restriction sites in the oligonucleotide sequence. These restriction sites may facilitate insertion of nucleic acid cassettes [i.e. PCR products] into an expression vector having the additional antibody sequences. In certain embodiments, restriction sites are designed to facilitate cloning of nucleic acid cassettes without introducing the outer nucleic acid sequences or by removing the original CDR or structural region sequences.

Nucleic acid cassettes may be cloned into any suitable expression server for part or all of the light or heavy chain sequence containing the generated target amino acid substitution. In accordance with the methods described in the invention, the nucleic acid cassette is cloned into a vector that allows production of part or all of the light or heavy chain sequences fused to all or only part of a viral capsid protein (i.e. of a fusion protein) displayed on the surface of a particle or cell. Although various beaker types are available and can be used for the practice of this invention, phagemid vectors are convenient because they can be constructed relatively easily and can be easily amplified. Phagemid vectors usually contain a variety of components, including promoters, signal sequences, phenotypic selection genes, replication sites, and other necessary components known to those skilled in the art.

In another example, in which a combination of a specific variant amino acid is to be expressed, the nucleic acid cassette contains a sequence that is capable of encoding all or part of the heavy or light chain variable domain, and is capable of encoding the combinations of the variant. of amino acid. For the production of antibodies that contain these variant amino acids or variant amino acid combinations, such as in a library, the nucleic acid cassette may be inserted into an expression vector containing the additional sequence of antibodies, for example, all or part of the variable domain region or constant of the antibody. light and heavy chain. These additional antibody sequences may also be fused to other nucleic acid sequences, such as the sequences that encode the components of capsideviral proteins and thus permit the production of a fusion protein.

Vectors

One aspect of the invention includes a nucleic acid-replicable expression vector comprising a sequence encoding a fusion gene, wherein the fusion gene encodes a fusion protein comprising a polypeptide contained in the CDR (such as an antibody variable domain), or a variable or constant domain of antibody fused to all or part of a viral capsid protein. Also included is a library of various replicable expression vectors comprising a variety of fusion genes encoding a variety of fusion proteins, including a variety of fusion polypeptides produced with multiple sequences, as described above. Vectors may include a variety of components and may be constructed to allow the variable domain of the antibody to be moved between different vectors and / or to provide fusion protein display in different formats.

Examples of vectors include phage vectors and phagemid vectors (which are illustrated herein, and described in greater detail above). A phage vector generally has an origin of phage replication allowing for application and phage particle formation. The phage is generally a filamentous bacteriophage, such as an M13 phage, fl, fd, Pf3 or a derivative thereof, or a lambdoid phage such as lambda, 21, phi80, phi81, 82, 424,434 and the like, or a derivative thereof.

Examples of viral capsid proteins include plll (sometimes also referred to as p3) infectivity protein, pVIII major dacapeptide protein, Soe (T4), Hoc (T4), bacteriophage lambda phage (pVI) gpD protein (phage filaments; J ImmunolMethods. December 1999; 10, 231 (1-2): 39-51), variants of the M13 bacteriophage capsid major protein (P8) (Protein Sci 9 April 2000; (4): 647 -54). Fusion protein may be displayed on the surface of a phage and suitable phage systems include phage helper systems M13K07, M13R408, M13-VCS, and Phi X 174, pJuFo (J Virol. August 2001; 75 (15): 7107- 13.v), hyperphage (Nat Biotechnol Jan 2001; 19 (1): 75-8). In certain embodiments, the phage helper is M13K07, and the proteins are the M13 phage gene III dacapsid proteins. In certain embodiments, the host cell is E. coli, and protease-deficient E. coli strains. Vectors, such as the fthl vector {Nucleic Acids Res. May 2001 15; 29 (10): E50-0) may be useful for expression of the fusion protein.

The expression vector may also have a secretion signal sequence fused to DNA encoding an oCDR-containing fusion polypeptide (e.g., each antibody subunit, or fragments thereof). This sequence is typically localized immediately to the 5 'of the fusion protein encoding gene, and therefore will be transcribed at the amino terminus of the fusion protein. However, in certain cases, it has been shown that the signal sequence is located at different positions than 5 'of the gene encoding the protein to be secreted. This target protein sequence is found all over the interior of the bacterial cell membrane. DNA encoding the signal sequence can be obtained as an endonuclease restriction fragment of a gene encoding a protein that has a signal sequence. Suitable signal sequences in prokaryotes can be obtained from genes encoding, for example, LamB or OmpF (Wonget al., Gene, 68: 1931 (1983), MalE, PhoA, and other genes. In one derealization example, a signal sequence The prokaryotic activity for this invention is the signal sequence of thermo-labile E. coli enterotoxin II (STII), as described by Chang et al., Gene 55: 189 (1987), and / or malB.

As indicated above, a vector also typically includes a promoter for directing expression of the fusion polypeptide. Promoter motors most commonly used in prokaryotic vectors include the lac Z promoter system, pho A (AP) alkaline phosphatase promoter, the 1PL temperature-sensitive promoter promoter. ), tac promoter, detriptophan promoter, bacteriophage T7 promoter. For general descriptions of the engines, see section 17 of Sambrook et al. Supra Although they are the most commonly used promoters, other suitable promoters may also be used.

The vector may also include other nucleic acid sequences, for example, sequences encoding gD markers, dec-Myc epitopes, polyhistidine markers, fluorescent proteins (e.g. GFP), or beta-galactosidase protein that may be useful in detecting or purification of fusion protein expressed on the cell surface or phage. Nucleic acid sequences encoding, for example, a gD marker also provide for the positive or negative selection of cells or viruses expressing fusion protein. In some embodiments, the gD marker is fused to an antibody variable domain that is not fused to a component of the viral capsid protein. Nucleic acid sequences encoding, for example, a polyhistidine tag are useful for identifying fusion proteins including antibody variable domains that bind to a specific antigen using the immunohistochemical technique. Markers useful for detecting antigen binding can be linked to either a non-fused antibody variable domain to a component of the viral capsid protein or to an antibody variable domain fused to a component of the viral capsid protein.

Another useful component of vectors used for this invention is the phenotypic selection genes. Specific phenotypic selection genes are genes that encode proteins that confer antibiotic resistance in host cells. For purposes of illustration, the gene conferring ampicillin resistance (ampr), and the tetracycline resistance gene (tetr) are readily employed for this purpose.

The vector may also include nucleic acid sequences containing unique restriction sites and stop codons that may be suppressed. Unique restriction sites are useful for transporting antibody variable domains between different vectors and expression systems, especially useful for the production of full length antibodies or antigen-binding fragments in cell cultures. Stop codons that can be suppressed are useful for controlling the expression level of the fusion protein and facilitating the purification of soluble antibody fragments. For example, an amber stop codon may be read as Glnem as a supE host to allow phage display, whereas a non-supE host is read as a stop codon to produce soluble antibody fragments without fusion to a capsid protein. Dophagus. These synthetic sequences may be fused to one or more antibody variable domains in the vector.

It is sometimes beneficial to use vector systems that allow nucleic acid coding for an antibody sequence of interest, for example, a CDR having variant amino acids, which can easily be removed from the vector system and placed in another vector system. For example, suitable restriction sites may be developed in a vector system to facilitate removal of the nucleic acid sequence that encodes an antibody or antibody variable domain with amino acid variants. The restriction sequences are usually chosen to be unique in the vectors to facilitate excision and binding in the new vector.

Antibodies or antibody variable domains can then be expressed by vectors without external fusion sequences, such as viral dacapid proteins or other marker sequence.

Between nucleic acids encoding the antibody constant or constant domain (gene 1) and the viral capsid protein component (gene 2), a DNA encoding a stop codon may be inserted, this termination codon includes UAG (Amber), SAU (Ocher) and UGA (Opal) (Microbiology, Davis et al., Harper & Row, New York, 1980, pp. 237, 245-47 and 374). The stop or stop codon expressed in a wild-type host cell results in the synthesis of nonprotein gene 1 protein produced by the attached gene 2. However, growth in a suppressing host cell results in the synthesis of detectable amounts of fused proteins. These suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al., BioTechniques 5: 376-379 (1987)). Any acceptable method can be used to place the stop codon in the fusion opolipeptide encoding mRNA.

The suppressable codon may be inserted between the first gene encoding an antibody variable or constant domain, and a second gene encoding at least a portion of the phage capsid protein. Alternatively, the suppressible termination codon may be inserted adjacent to the fusion site by substituting the last triad of amino acid in the variable domain of the antibody or first amino acid in the phage capsid protein. The terminating codon that may be deleted may be located at or after the C-terminal end of a dimerization domain. When the plasmid containing the suppressable codon is cultured in a suppressor host cell, it results in the detectable production of a fusion polypeptide containing the capsid polypeptide and aprotein. When the plasmid is cultured in a non-suppressing host cell, the antibody variable domain is substantially synthesized without fusion of the phagemid capsid protein to the terminator inserted into the UAG, UAA or UGA suppressor triad. In non-suppressing cells the antibody variable domain is synthesized by the host cell, due to the absence of fusion of the dacapsid fire protein with another membrane-anchored protein of this host cell.

In some embodiments, the CDR being diversified (randomized) may have a stop codon designed in the template sequence (referred to herein as a "stop template"). This feature successfully provides for the detection and selection of diversified sequences based on stop codon (s) detection in the template sequence due to the incorporation of the oligonucleotide (s) comprising the sequence (s) for variant amino acids. interest. This feature is further illustrated in the following Examples.

The variable or constant domain of the antibody light and heavy chain may also be fused to an additional peptide sequence, providing the interaction of one or more fusion polypeptides on the surface of the viral particle or cell. These peptide sequences are referred to herein as "dimerization domains". Dimerization domains may include at least one or more dimerization sequences or at least one sequence that includes a cysteine residue or both. Suitable dimerization sequences include that of proteins having an amphipathic alpha helix in which hydrophobic residues are regularly spaced and allow the formation of a dimer by the interaction of hydrophobic residues of each protein; such proteins and protein portions include, for example, deleucine zipper regions. Dimerization domains may also include one or more cysteine residues (for example, as provided by the inclusion of an antibody hinge region sequence within the dimerization domain). Cysteine residues may provide dimerization by formation of one or more disulfide bonds. In one example, in which the stop codon is present after the dimerization domain, the dimerization domain comprises at least one cysteine residue. In some embodiments, the dimerization domains are located between the variable or constant antibody domain and the components of the viral dacapid protein. In some cases, the vector encodes a single antibody-phage polypeptide into a single chain containing, for example, both of the two. heavy and light variable regions fused to a capsid protein. In these cases, the ovector is considered to be "monocistronic", expressing a transcription under the control of a specific promoter. For example, a vector may utilize a promoter (such as alkaline phosphatase (AP) or Tac promoter) to drive expression of a monocistronic sequence encoding the V1 and Vh domains with a linker peptide between the V1 and Vh domains. This cistronic sequence may be attached to the 5 'end of a signal sequence (such as a malE or thermo labile enterotoxin II (STII) signal sequence) and, at its 3' end, the complete viral capsid protein or match this (such as the bacteriophage plll protein). The fusion polypeptide encoded by a vector of this embodiment example is referred to as "ScFv-plH". In some embodiments, a vector may further comprise a sequence encoding a dimerization domain (such as a leucine zipper) at its 3 'end, between the second variable domain sequence (e.g., Vh) and the viral capsid protein sequence. Fusion polypeptides comprising the dimerization domain are capable of dimerizing to form a complex of two scFv polypeptides (referred to herein as "(ScFv) 2-plll").

In other cases, heavy and light chain variable regions may be expressed in separate polypeptides, thus using a "bicistronic" vector, allowing the expression of separate transcripts. In these vectors, a suitable promoter, such as the Ptac or PhoA promoter, is used to direct expression of the bicistronic message. A first cistron encoding, for example, a light chain variable ominium, can be connected to the 5 'end of a signal sequence, such as a malE or E. colie thermo labile enterotoxin II (STII) sequence at the 3'end nucleic acid sequence encoding a tag sequence, such as a gD tag. A second citron, encoding, for example, a variable domain and a CH1 heavy chain constant domain, is linked at its 5 'end to a signal sequence, such as a E. coli thermo labile enterotoxin II (STII) signal sequence. and at the 3 'end a protein of the complete viral capsid or at the end thereof.

In one example of a vector providing a bicistronic message for displaying F (ab ') 2-plll, a suitable promoter, such as the Ptac promoter or PhoA (AP) promoter, drives expression of a first citrus encoding the light chain variable domain and a constant domain operably linked to the 5 'end of a signal sequence, such as a malE signal sequence or therelabile enterotoxin II (STII) daE. coli and at the 3 'end a nucleic acid sequence encoding a labeling sequence such as gD marker. The second citron encodes, for example, a variable and constant heavy chain domain operably linked at the 5 'end to a signal sequence, such as a E. coli thermo labile enterotoxin II (STII) signal sequence and at the 3' end has a dimerization domain comprising the IgG hinge region and a leucine zipper sequence followed by at least a portion of the viral capsid protein.

Fusion Polypeptide Display

Fusion polypeptides of a CDR-containing polypeptide (for example, an antibody variable domain) may be displayed on the surface of a cell, virus, or phagemid particle in a variety of forms. These forms include single chain Fv (scFv) fragments, fragment F (ab) polyvalent forms of these fragments. For example, multivalent forms include one of ScFv, Fab, or F (ab '), referred to herein as (ScFv) 2, F (ab) 2 and F (ab') 2, respectively. Multivalent display forms are advantageous in some contexts, in part because they have more than one antigen-deletion site that generally results in the identification of lower affinity clones and also allows for more efficient screening of rare clones during the selection process.

Methods for displaying fusion polypeptides comprising antibody fragments on the surface of the bacteriophage are well known in the state of the art, for example, as described in WO 92/01047 and herein. Other patent publications WO 92/20791, WO 93/06213, WO 93/11236 and WO 93/19172 describe methods and are all incorporated herein by reference. Other publications have demonstrated the identification of artificially rearranged V-gene antibodies against a variety of antigens displayed on the surface of a phage (as, for example, in HRHoogenboom & G. Winter, J. Mol. Biol. 227 381-388 (1992); and such as disclosed in WO 93/06213 and WO 93/11236 ).

When a vector is constructed for display in an scFv form, it includes nucleic acid sequences encoding an antibody light chain variable domain and an antibody heavy chain domain. Typically, the nucleic acid sequence encoding a chain variable domain The heavy antibody antibody is fused to a component of the viral dacapsid protein. One or both antibody variable domains may have variant amino acids in at least one CDR region. The nucleic acid sequence encoding the light chain variable domain is connected to the nucleic acid sequence encoding the heavy chain variable domain by a nucleic acid sequence encoding a ligand polypeptide. Binding opolipeptide usually contains about 5 to 15 amino acids. Optionally, other coding sequences, e.g., useful markers for purification or detection, may be fused to the 3 'end of the nucleic acid sequence encoding the light chain variable domain or heavy chain variable domain. or both.

When a vector is assembled for display F (ab), it includes nucleic acid sequences that encode antibody variable domains and antibody constant domains. A nucleic acid encoding a light chain variable domain is joined to a nucleic acid sequence encoding a light chain constant domain. A nucleic acid sequence that encodes an antibody heavy chain variable domain is joined to a nucleic acid sequence encoding a heavy chain constant CH1 domain. Typically, the nucleic acid sequence encoding variable domain and heavy chain constants are joined to a nucleic acid sequence encoding all or part of a viral envelope protein. Either antibody light or heavy chain variable domains may have variant amino acids in at least one CDR. In some embodiments, variable domain and heavy chain content are expressed as a fusion with at least a portion of a viral envelope protein, and variable domain and light chain content are expressed separately from a heavy chain fusion viral envelope protein. The heavy weight chains are associated with each other and may be by covalent or non-covalent bonding. Optionally, other sequences encoding polypeptide expression useful for purification and detection, for example, may be joined at the 3 'end of the nucleic acid sequence encoding the antibody light chain constant domain or antibody heavy chain constant domain or both.

In some embodiments, a bivalent molecule, for example an F (ab) 2 or F (ab ') 2 dimer, is used to display antibody fragments with variant amino acid substitutions on the surface of a particle. F (ab ') 2 dimers have generally been found to have the same affinity as F (ab) dimers in a liquid phase antigen binding assay, but the dissociation rate of F (ab') 2 is lower due to higher greed. Therefore, the bivalent form (e.g. F (ab ') 2) is a particularly useful form as it can allow identification of lower affinity clones and also allows more efficient screening of rare clones during the selection process.

Introduction Of Vectors In Host Cells

Vectors constructed as described in accordance with the invention are introduced into a host cell for amplification and / or expression. Vectors can be introduced into host cells using normal modification methods, including electroporation, calcium phosphate precipitation and the like. If the vector is an infectious particle, such as a virus, the vector itself provides entry into the host cell. Transfection of host cells containing an replicable expression vector encoding a fusion gene and phage particle production according to standard procedures provides phage particles in which the fusion protein is displayed on the surface of the phage particle.

Replicable expression vectors are introduced into host cells using a variety of methods. In one embodiment, vectors may be introduced into cells using electroporation as described in WO / 00106717. Cells are cultured in standard culture broth, optionally for about 6 to 48 hours (or until reaching an OD60 = 0.6-0.8) at about 37 ° C, and then the broth is centrifuged and the supernatant removed. (e.g., by decantation). In some embodiments, initial purification includes resuspension of the cell pellet in a buffer solution (e.g. 1.0 mM HEPES pH 7.4), followed by re-centrifugation and removal of the supernatant. The resulting cell pellet is resuspended in glycerol diluent (e.g., 5-20% v / v) and centrifuged again to form a cell pellet and the supernatant removed. The final concentration is obtained by cell resuspension of the cell pellet in water or diluted glycerin at the desired concentration.

In certain embodiments, the recipient cell is an electroporation competent E. coli strain of the present invention, which is E. colicepa SS320 (Sidhu et al., Methods. Enzymol. (2000), 328: 333-363) . Strain SS320 was prepared by crossing with MCI061 XL1-BLUE cells under conditions sufficient to transfer fertility episode (plasmid F ') or XL1-BLUE into MC1061 cells. The SS320 strain was deposited under the American Type Culture Collection (ATCC) number, 10801 University Boulevard, Manassas USA, on June 18, 1998 and under Accession No. 98795. Any F 'episode allowing phage replication in the strain can be used in the invention. Suitable episodes are available from the ATCC deposited dishes or are commercially available (CJ236, CSH18, FHD, JM101, JM103, JM105, JM107, JM109, JM110), KS1000, XL1-blue, 71-18 and others).

Using higher concentrations of DNA during electroporation (about 10X) increases the modification efficiency and the amount of modified DNA in the host cells. The use of high cell concentrations (about 10X) also increases efficiency. The larger amount of transferred DNA produces larger libraries with greater diversity and represents a large number of single members of a combinatorial library. Transformed cells are usually selected for growth in medium containing antibiotics.

Selection (Screening) and Binders Tracking for Choice Targets

The use of phage display for the identification of ligands for the target antigen, with their various permutations and variations in methodology, is well established in the state of the art. One approach involves constructing a family of replicable variant vectors containing a transcriptional regulatory element operably linked to a gene encoding a fusion polypeptide, modifying suitable host cells, and culturing transformed cells to form phage particles displaying the fusion polypeptide on the phage surface, followed by a process which involves selecting or sorting by exposing the recombinant phage particles with a target antigen such that at least a portion of the particle population binds to the target in order to enhance and enrich the particle-binding subsets of particles. that do not care about in the selection process. The selected pool may be amplified by infection in host cells, such as XLI-Blue cells, for another screening step on the same target with the same or different restriction. The variant pool is then screened against the target antigen to identify novel high affinity ligand proteins. These novel high affinity binding proteins may be useful as therapeutic agents as agonists or antagonists, and / or as diagnostic and research reagents.

Fusion polypeptides such as antibody variable domains comprising variant amino acids may be expressed on the surface of a phage, phagemid particle or cell and then selected and / or screened by the ability of members of the fusion dopolipeptide group to bind to a target antigen. which is typically the antigen of interest. Selection procedures for target ligands may also be included by selecting on a generic protein having antibody variable domain affinity such as the L protein or a specific antibody-binding antibody marker or antibody fragments displayed on the phage that may be used. to enrich library members that display correctly folded antibody fragments (fusion polypeptides).

Target proteins such as DR5 and HER-2 receptors may be isolated from natural sources or prepared by recombinant methods by known procedures in the art. Human and murine DR5 sequences are provided in Table 1. The sequence and preparation of HER-2 2 ECD has been described in Franklin MC. Carey KD. VajdosFF. Leahy DJ. from Vos AM. Sliwkowski MX, Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex, Cancer Cell. 5 (4): 317-28, 2004. Amino acid sequence of an extracellular domain 23-646 of HER-2 is provided in Protein DataBank Record 1H78 (2004). Target antigens may include a number of therapy molecules of interest.

A variety of affinity selection (screening) strategies can be used. An example is a method of solid support screening or plate screening or immobilized target screening. Another example is the solution binding method. For the solid support screening method, the target protein can be fixed to an appropriate solid or semi-solid matrix. Such matrices are known in the art such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic polymers, hydroxyl methacrylate gels, polyacrylic epolimethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Affixation of the target protein to the matrix can be accomplished by the methods described in Methods in Enzymology, vol. 44 (1976) or another known technique in the state of the art.

Following fixation of the target antigen on the matrix, the immobilized target is exposed to the library expressing the fusion polypeptides under conditions suitable for the binding of at least a subset of the phage particle with the immobilized target antigen. Typically, conditions including pH, ionic strength, temperature and the like will mimic physiological conditions. Particles bound ("ligands") to the immobilized target are separated by washing off particles that do not bind to the target. Washing conditions may be adjusted to result in the removal of all but the high affinity binders. Binders can be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using a wild-type binder (e.g., excess antigen targeting), altering pH and / or ionic strength, and other methods known in the art. Selection of binders typically involves eluting an affinity matrix with a material suitable for elution such as acid such as 0.1M HCl or binders. Elution with increasing binder concentration could elucidate increased affinity displayed binder molecules.

Binders can be isolated and then re-amplified into suitable host cells by infecting the cells with the viral particles that are ligands (and phage helpers if necessary, for example when the viral particle is a phagemid particle) and the host cells are then cultured under conditions suitable for amplification of particles exhibiting the desired fusion polypeptide. Phage particles are collected and the selection process is repeated one or more times until the target antigen ligands are enriched. Any number of selection or screening steps may be used. One of the selection or screening processes may involve the isolation of ligands which bind to a generic affinity protein such as, for example, Lou protein, an antibody to a marker polypeptide present in the displayed polypeptide, such as antibodies to the gD protein or poly marker. -histidine. Counter-selection may be included in one or more steps of selection or screening to isolate ligands that also exhibit unwanted binding to one or more non-target antigens. One aspect of the invention involves selection against inventive libraries using a novel selection method which is termed "deletion method". solution". The invention is solution phase screening much more efficiently than conventional solution screening methods. The solution binding method can be used to find original ligands from a randomized library or by identifying improved ligands in a library that has been designed to improve the affinity of a particular ligand clone or group of clones. The method comprises exposing a variety of polypeptides, such as phage-exposed polypeptides or phagemid particles (library), with a labeled target antigen or fused to a marker molecule. The marker may be biotin or other specific binding molecules available for this purpose. The stringency of the solution phase may vary using decreasing concentrations of labeled target antigen in the first solution phase. To further enhance stringency, the first solution binding phase may be followed by a second solution phase with high concentration of unlabeled target antigen after initial binding with the labeled antigen in the first phase dissolution. Typically, a 100 to 1000-fold excess of unlabeled target antigen is used in the second phase (if included). The incubation period of the first solution phase may range from a few minutes to one or two hours or more to achieve equilibrium. Using a short time period in this first binding phase can influence or select ligands that have rapid dissociation velocity. The incubation time and temperature in the second phase can be modified to increase strain. This provides a selection influence for ligands that have slow target slow-off rate. After exposure of a variety of polypeptides (displayed on phage / phagemid particles) to a target antigen, the phage or particles Defagomides that are bound to the tagged target antigens are separated from the non-bound phages. The target particle mixture in the deligant solution phase is isolated by exposing it to the labeled target molecule and allowing these to bind to the molecule that is bound to the labeled target molecule for a short time (e.g., 2 to 5 minutes). The initial concentration of labeled target antigen may range from about 0.1 nM to about 1000 nM.

The bound particles are eluted and can be propagated to the next sorting step. In certain embodiments, various screening steps are performed using lower and lower concentrations of labeled target antigen at each screening step.

For example, an initial classification or selection using about 100 to 250 nM of labeled target antigen should be sufficient to capture a wide range of affinities, although this factor may be determined empirically and / or adapted to the technician's desire. In the second selection step, about 25 to 100 nM of labeled target antigen may be used. In the third selection step, about 0.1 nM to 25 nM of labeled target antigen may be used. For example, to improve the 100 nM affinity of a binder, it may be desirable to start with 20 nM and then progressively increase from 1 to 5 nM of labeled target antigen and then followed by an even lower concentration as around 0, 1 nM of labeled target antigen.

The conventional screening solution involves the use of spheres such as streptoavidin coated spheres, which are too heavy to use and often result in very low phagoligant recovery efficiency. The conventional ball sorting solution takes much longer than 2-5 minutes and is less viable to adapt to the high automated production that the invention describes above.

As described herein, combinations of a solid support and solution screening methods can be advantageously used to isolate binders with desired characteristics. Following selection / screening of the target antigen for a few steps, screening of individual clones from a pool is usually performed to identify specific ligands of desired properties / characteristics. In some embodiments, the sorting process is performed by automated systems to enable high throughput screening of candidate libraries.

Two main screening methods are described below. However, other methods known in the art may also be used in the methods of the invention. The first screening method includes a phage ELISA assay with immobilized target antigen, which allows the identification of a specific binding clone from a non-binding clone. Specificity can be determined by simultaneous analysis of clone in target-coated wells and BSA or wells coated with other proteins that are not the target proteins. This assay is automated for high throughput selection.

An exemplary embodiment provides a method for selecting an antibody variable domain that binds to a specific target antigen from an antibody variable domain library, generating a replicable expression vector library comprising a variety of polypeptides; and exposing the library with a target antigen and at least one antigen other than the target antigen under conditions suitable for binding; splitting the binding polypeptides into the non-binding library; eidentifying ligands that bind to the target antigen and those that do not bind antigen other than the target antigen; and eluting the target antigen ligands, and amplifying replicable expression vectors containing the ligand polypeptides that bind to a specific antigen.

The second screening assay is an affinity screening assay provided to separate high affinity clones from low affinity clones in a high throughput manner. In the assay, each clone is tested with and without the first incubation with the specific concentration target antigen for a period of time (eg 30 to 60 minutes) before application to antigen coated wells (eg 5 to 15 minutes). minutes). Then bound phage are measured by the usual phage ELISA method, for example using HRP-conjugated anti-M13. The binding signal ratio of the two wells, one that was preincubated with antigen and the other and thus not preincubated with target antigen is an indication of affinity. Selecting the target antigen concentration for the first incubation depends on the affinity range of interest. For example, if affinity binders greater than 10 nM are desired, then 100 nM target antigen is often used in the first incubation. Since ligands are found from a screening (selection) step, these clones can be screened with a screening affinity assay to identify the highest affinity ligands.

Combinations of some of the screening / selection methods described above can be performed with the screening methods. For example, in one example, the linker polypeptides are first selected by binding to the immobilized target antigen. Binder polypeptides that bind to the immobilized target antigen can then be amplified and screened for binding to the target antigen and for the absence of binding to antigens other than the target antigens. Binder polypeptides that specifically bind to the target antigen are then amplified. These linker polypeptides can then be selected by high affinity by exposure with a target antigen concentration labeled to form a complex, at a range of labeled antigens from about 0.1 nM to 1000 nM, the complexes are isolated by exposure with an agent that binds to the marker on the target antigen. Binding polypeptides are eluted from the labeled target antigens and optionally, the selection steps are repeated using always a lower concentration of labeled target antigen at each step. High affinity linker polypeptides isolated using this deletion method can then be screened for high affinity using a variety of methods known in the art, some of which are described herein.

These methods can localize high affinity clones without having to perform long and complex competition affinity assays on a large number of clones. The intensive aspect of performing complex trials of many clones is often a major obstacle to finding better clones by selection. This method is especially useful in native affinity improvement in which various binders with similar affinities can be recovered from the selection process. Different clones may have the expression / display efficiency on phage or very different phagemid particles. These more highly expressed clones have a better chance of recovery. That is, selection may be induced by the display or expression levels of the variants. The solution binder classification method of the invention can improve the screening process for locating high affinity binders. This method is an affinity tracking assay that provides a significant advantage in terms of screening for better binders quickly and easily.

Antibodies or antigen-binding fragments may still be selected for functional activity, for example, for example, agonist or antagonist activity. For example, anti-HER-2 antibodies may be selected by the ability to inhibit tyrosine phosphorylation in HER-2 and proliferation of cancer cells or to induce apoptosis in cancer cells. Assays to identify and measure such activities are described, for example, in WO98 / 17797. In addition, anti-DR5 antibodies may be selected for their ability to induce apoptosis in cancer cells and / or inhibit the function of inflammatory cells. In other embodiments, anti-DR5 antibodies are selected for the ability to compete with DR5 for Appo-2L binding. In other embodiments, human anti-DR5 antibodies are selected for murine DR5 and / or DR5 binding of cynomolgus monkeys. to determine biological activity can be performed by methods known in the art, such as DNA fragmentation (see, for example, Marsters et al., Curr. Biology, 6: 1669 (1996)), caspase inactivation, binding to DR5 (see, for example, WO98 / 51793, published November 19, 1998. The level of apoptosis can be measured by identifying cytoplasm condensation, lost plasma membrane micro villi, nucleus segmentation, chromosomal DNA degradation or loss of mitochondrial function This activity can be determined and measured, for example, by cell viability assays (such as the Alamar blue assay or MTT), FACS analysis, caspase activation, DNA fragmentation (see, for example, Nicoletti et al., J. Immunoi Methods, 139: 271-279 (1991), poly-ADP ribose epimerimerase, PARP cleavage and known assays in the state of the art.

In one embodiment, such as the apoptosis assay involves performing twice the serial dilutions of the standard control and anti-DR5 antibody in 96-well culture plates. Apo-2 ligand (amino acids 114-281, described in PCT US 00/17579) is tested for breakdown effects. Colo-205 (20,000 well cells) with human colon carcinoma (ATCC) cells are seeded in 96 well plates. The plates are incubated at 37 ° C for 24 hours. AlamazBlue (Trek Diagnostic Systems, Inc.) is added to the wells within the last 3 to 24 hours of incubation time. Influorescence is read using a 96-well plate fluorometer using 530 nm excitation wavelength and 590 nm emission wavelength. Results are expressed as relative fluorescence units (RFU).

Following identification of the ligands by binding to the target antigen, and / or functional assays of nucleic acids may be extracted. The extracted DNA can then be used directly to transform E. coli host cells or alternatively, the coding sequences can be amplified, for example, using PCR with suitable primers, sequenced by any typical sequencing method. The ligand variable domain DNA can be digested by restriction enzyme and then inserted into a vector for protein expression.

Populations comprising polypeptides having CDR (s) restricted diversity sequences according to the methods of the invention may be used to isolate ligands against a variety of targets, including those listed in Figures 11, 15, 21A and 24A. These ligands may comprise one or more variant CDRs spanning several sequences generated using restricted codons. In some embodiments, a variant CDR is CDRH3 comprising sequence diversity generated by amino acid substitution with a restricted codon set and / or amino acid insertions resulting from CDRH3 of varying lengths. Illustrative ligonucleotides useful for the generation of invention fusion polypeptides include those of the invention. 9A-D, Figures 20A-L and Figure 23. One or more variant CDRs may be combined. In some embodiments, only CDRH3 is diverse. In other examples, two or more heavy chain CDRs, including CDRH3, are variants. In other embodiments, one or more heavy chain CDRs, excluding CDRH3, are variant. In some embodiments, at least one heavy chain and at least one light chain CDR are variants. In some performing embodiments, at least one, two, three, four, five, or all H1, H2, H3, L1, L2 and L3 are variants.

In some cases, it may be beneficial to combine one or more diverse light chain CDRs with the novel ligands isolated from a polypeptide population that includes one or more diverse heavy chain CDRs. This process can be referred to as a step-2 process. An example of a step-2 process first comprises determining deligants (generally lower affinity binders) within one or more libraries generated by randomizing one or more CDRs, in which the randomized CDRs in each library are different, or, in the case of the library. Even though randomized CDRser, it is randomized to generate different sequences. Binders of a heavy chain library can then be randomized to a variety of light chain CDRs, for example, by the mutagenesis technique, such as the Kunkel technique, or by cloning (cutting and pasting (for example, by ligating different sequences). CDR joins)) The new decade-old light chain heavy ligand library that has only one light chain fixation. The pool can then be further ranked against one or more targets to identify binders that have increased affinity. Binders (eg, low affinity binders) obtained from H1 / H2 / H3 screening can, for example, be fused to a diversity. L1 / L2 / L3 libraries to replace the fixed original L1 / L2 / L3, in which the new libraries are then further selected against a target of interest to obtain another set of ligands (eg, high affinity ligands). New antibody sequences that exhibit higher binding affinity can be identified by a variety of target antigens.

In some embodiments, libraries comprising polypeptides of the invention are subjected to a variety of screening steps, each step including screening comprising the doligant exposure obtained from the previous step with a distinct target antigen (s). ) of the target antigen (s) from the previous step (s). Preferably, but not necessarily, the target antigens are homologous in sequence, for example, members of a family of related but distinct polypeptides, such as, but not limited to, cytokines (eg, interferon alpha subtypes). Library Production Understanding Polypeptides Containing CDR

Variant

Variant CDR polypeptide libraries may be produced by mutation of a solvent accessible position and / or highly diverse into at least one CDR of an antibody variable domain.

Some or all of the CDRs may be mutated using the methods of the invention. In some embodiments, it may be preferable to generate multiple antibody libraries by mutating at positions of CDRH1, CDRH2 and CDRH3 to form a single library or by mutating depositions into CDRL3 and CDRH3 to form a single library or position-changing CDRL3 and CDRH1, CDRH2 and CDRH3 to form a single library.

An antibody variable domain library can be produced, for example, by mutations in the solvent accessible and / or highly diverse position of CDRH1, CDRH2 and CDRH3. Another library can be generated with mutations in CDRL1, CDRL2, and CDRL3. These libraries can also be used in conjunction with others to generate desired affinity binders. For example, after one or more steps of selecting heavy chain libraries for binding to a target antigen, a light chain library may be replaced in the heavy chain ligand population by additional deletion steps to increase ligand affinity. In one embodiment, a library is created by substituting the original amino acids with a limited set of variant amino acids in the CDRH1, CDRH2, and / or CDRH3 region of the heavy chain sequence variable region and / or the light chain sequence variable region CDRL3 region. According to the invention, this library may include a variety of antibody sequences, in which sequence variance is primarily in the CDRH3 region of the heavy decade sequence.

In one aspect, the library is created in the context of the humanized 4D5 antibody sequence, or the amino acid sequence of the humanized 4D5 antibody structural region. In certain embodiments, the library is created by replacing at least residues 28 and 30-33 of CDRH1, residues 50, 52-54, 56 and 58 of CDRH2, residues 95, 96, 97, 98.99 100, 100a, 100b and 100c of CDRH3, residues 91-94 and 96 of CDRL3 as set of amino acids set forth as shown in Figure 8 for the "YSGR-A" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52-54, 56 and 58, residues 95, 96, 97, 98, 99 , 100,100a, 100b and 100c of CDRH3, amino acid set CDRL3 residues 91-94 and 96 set forth as shown in Figure 8 for the "YSGR-B" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52-54, 56 and 58, residues 95, 96, 97, 98, 99 , 100,100a, 100b and 100c of CDRH3, and amino acid set CDRL3 residues 91-94 and 96 set forth as shown in Figure 8 for the "YSGR-C" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52-54, 56 and 58, residues 95, 96, 97, 98, 99 100,100a, 100b and 100c of CDRH3, CDRL3 residues 91-94, 96 with the amino acid set established as shown in Figure 8 for the "YSGR-D" library. Amino acid positions 100b and 100c may have a different alphabetic identifier depending on the length of CDRH3, but these positions are the last two positions of CDRH3 before position 101. Examples of suitable oligonucleotide sequences include, but are not limited to, those listed in Figures 9A -D and may be determined by the subject matter expert according to the criteria described herein.

In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set amino acids set as shown in Figure 19A for the "SAH3" library. In certain embodiments, the library is created by substituting at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 19A for the "SCH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set of amino acids established as shown in Figure 19A for the "SFH3" library. In certain embodiments, the library is created by substituting at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 19A for the "SGH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set of amino acids established as shown in Figure 19A for the "SIH3" library. In certain exemplary embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110m , amino acid pool residues 91-94 and 96 set forth as shown in Figure 19A for the "SLH3" library. As the length of CDRH3 varies, the last two positions before position 101 may have different alphabetic identifiers depending on the length of CDRH3, but these positions are the last two positions of CDRH3 before position 101. Examples Suitable oligonucleotide sequences include, but are not limited to a, those listed in Figures 20A-20L, and may be determined by the subject matter expert in accordance with the criteria described herein.

In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50.52, 53, 54, 56 and 58, CDRH3 residues 95-110, residues 91-94 and 96 of CDRL3 with the amino acid set set as shown in Figure 19B for the "SNH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 19B for the "SPH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set amino acids set as shown in Figure 19B for the "SRH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 19B for the "STH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set amino acids set as shown in Figure 19B for the "SWH3" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 19B for the "SYH3" library. As the length of CDRH3 varies, the last two positions before position 101 may have different alphabetic identifiers depending on the length of CDRH3, but these positions are the last two positions of CDRH3 before position 101. Examples Suitable oligonucleotide sequences include, but are not limited to a, those listed in Figures 20A-20L, and may be determined by the subject matter expert in accordance with the criteria described herein.

In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set amino acids set as shown in Figure 22 for the "SY" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the minoacid set set as shown in Figure 22 for the "SW" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110, CDRL3 residues 91-94 and 96 with the set amino acids set as shown in Figure 22 for the "SR" library. In certain embodiments, the library is created by replacing at least CDRH1 residues 28 and 30-33, CDRH2 residues 50, 52, 53, 54, 56 and 58, CDRH3 residues 95-110 m, CDRL3 residues 91-94 and 96 with the amino acid set set as shown in Figure 22 for the "SF" library. Just as the length of CDRH3 varies, the last two positions before position 101 may have different alphabetic identifiers depending on the length of CDRH3, but these positions are the last two positions of CDRH3 before position 101. Examples of suitable oligonucleotide sequences include, but are not limited to those listed in Figure 23, and may be determined by the subject matter specialist according to the criteria described herein.

In certain embodiments, a library is created by combining with other libraries. In one embodiment, the "SXH3" library as used herein comprises the libraries, SAH3, SCH3, SFH3, SGH3, SIH3, SLH3, SNH3, SPH3, SRH3, STH3, SWH3 and SYH3. In another example, the "SX-surface" library comprises the "SY", "SW", "SR" and "SF" libraries.

In another example, different CDRH3 designs are used to isolate high affinity binders and to isolate binders for a variety of epitopes. For diversity in CDRH3, several libraries can be constructed separately with different lengths of H3 and then combined for selection of ligands for target antigens. The length range of CDRH3 generated in this library can be 10-21,11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 19-21 20-21amino acids, although different lengths thereof may also be generated. Diversity can also be generated in CDRH1 and CDRH2, as indicated above. In one example embodiment of a library, diversity H1 and H2 is generated using the oligonucleotides illustrated in Figures 9A-D, L-20A and Figure 23. Other oligonucleotides with sequence variations may also be used. Oligonucleotides may be used alone or grouped in a variety of combinations depending upon the practical function of the practitioner's need and desires. In some embodiment examples, randomized positions on the heavy chain CDRs include those listed in Figures 8, 9, 11,15, 19, 21, 22, 23, and 24.

Multiple libraries can be assembled and ordered using solid support and solution selection methods as described herein. Multiple classification strategies can be employed. For example, one variation involves classification on the solid phase bound target, followed by screening for a marker that may be present on the fusion polypeptide (e.g., anti-gD marker) and then another classification on the solid phase bound target. Alternatively, libraries may be first classified into a target bound to a solid surface, the eluted ligands are then ordered using a liquid phase binding with decreased target antigen concentrations. Using combinations of different screening methods provides minimization of selection of highly expressed sequences only and provides selection of a high number of clones of different affinities.

From ligands isolated from the library pool as described above, it has been found that, in some cases, affinity can be improved by providing limited diversity to the light chain. Light chain diversity can be, but is not necessarily generated by variations in amino acid positions 91-96 in CDRL3 or a subset thereof. In one example, the randomized positions are those listed in Figures 8, 9, 11,15, 19, 21, 22, 23, and 24.

High affinity binders isolated from libraries of these embodiments are easily produced in bacteria and cultured in high yielding eukaryotic cells. Vectors can be designed to easily remove sequences such as the gD marker, the viral capsid protein component sequence, and / or to grow in constant region sequences to provide production of full-length antibodies or fragments binding to the antigen on self-care. Any combination of codon sets and CDRs may be diversified according to the methods of the invention.

Host Cell Vectors and Recombinant Methods

For recombinant production of an antibody of the invention, encoding nucleic acids are isolated and inserted into a replicable vector for cloning (DNA amplification) or expression. By encoding the DNA the antibody is easily isolated and sequenced using standard procedures (for example, by the use of deoligonucleotide probes that are capable of specifically binding to antibody light and heavy chain genodecoders). Many vectors are available. The choice of vector depends in part on the host cell that will be used. Generally preferred host cells include, but are not limited to, prokaryotic or eukaryotic (generally demamiferous) cells.

Antibody Production Using Prokaryotic Host Cells

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibody of the invention may be obtained using standard recombination techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides may be synthesized using polynucleotide synthesizers or PCR techniques. Once obtained, the polypeptide coding sequences are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors which are available and known in the art can be used for the purposes of the present invention. Selection of an appropriate vector will depend primarily on the size of the nucleic acids to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains several components, depending on their function (amplification or expression of heterologous polynucleotides, or both), and their compatibility with the particular host cell in which they reside. Vector components generally include, but are not limited to: a replication source, a selection marker gene, a promoter, a ribosome deletion site (RBS), a signal sequence, heterologous acidic insertion, and a transcription termination sequence. .

Generally, recombinant plasmid vectors containing control and replicon sequences that are derived from host cell compatible species are used in connection with these hosts. The receptor normally conducts as main chain components a breeding site origin as well as tag sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using a pBR322, a plasmid obtained from an E. coli species. pBR322 which has the genes that confer resistance to ampicillin (Amp) and tetracycline (Tet) and thus provides means for identifying transformed cells. PBR322, derivatives thereof, or other microbial or bacteriophage plasmids may also contain, or be modified by, promoters that may be used by the organismomicrobial for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of specific antibodies are described in detail in Carter et al, US Patent 5,648,237.

In addition, phage vectors that contain replica control sequences that are compatible with the host microorganism can be used as transformation vectors in connection with such hosts. recombinant vector manufacture that can be used to transform susceptible host cells such as E. coli LE392.

The expression vector of the invention may include two or more citron-enhancing pairs encoding each polypeptide component. The engine is an untranslated sequence of regulation located upstream (5 ') of a citrus that modulates its expression. Prokaryote promoters generally fall into two classes, the inducible and the constitutive. Inducible promoters are promoters that initiate increased levels of citrus transcription under their control in response to changes in culture condition, for example, the presence or absence of a nutrient or change. temperature.

A large amount of promoters are recognized by a number of potential host cells. The selected promoter may be operably linked to a light-chain or heavy-chain codon DNA by removing the promoter from the source DNA by restriction enzymes and inserting the isolated promoter sequence into the vector of the invention. Both native promoter sequence and many heterologous promoters can be used to direct amplification, as they generally allow for greater transcription and higher yields of the expressed target gene as compared to the native target promoter polypeptide.

Promoters suitable for use with prokaryotic hosts include the phoA promoter, lactose and 8-lactamase promoter systems, tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that are bacterially functional (such as other known phage or bacterial promoters) are also appropriate. Their nucleotide sequences have been published to enable those skilled in the art to operably link them to target light and heavy chain transcription units (Siebenlist et al, Cell, 20: 269 (1980)) using linkers or adapters to provide any restriction sites. needed.

In one aspect of the invention, each citron in the recombinant vector comprises a component of the secretion signal sequence that translocates expressed polypeptides across a membrane. Generally, the signal sequence may be a component of the vector or may be a part of the target polypeptide DNA that is inserted into the vector. Secretion signaling sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e. cleaved by a signaling peptidase I) by the host cell. For prokaryotic host cells that do not recognize and process native signaling sequences for heterologous polypeptides, the signaling sequence is replaced by a selected prokaryotic signaling sequence, for example from the group consisting of alkaline dephosphatase, penicillinase, Ipp or aocalor stable enterotoxin II (STII) leaders. ), LamB, PhoE, PelB, OmpA and MBP. In one example, the signal sequence used in both cistrons of the expression system is a STII signal sequence or variant thereof. In another aspect, the production of immunoglobulins can occur in the host cell cytoplasm, and thus does not require the presence of signal sequences of secretion within each citron. In this regard, light and heavy immunoglobulin chains are expressed in double and pooled to form functional immunoglobulins in the cytoplasm. Certain host cell lines (e.g., E. coli trxB strains) provide cytoplasmic conditions that favor the formation of disulfide bridges, thereby allowing the hinge and proper assembly of the expressed protein subunits. Proba and Plukthun, Gene, 159: 203 (1995).

The present invention provides an expression system in which quantitative ratio of the expressed polypeptide component can be modulated so as to maximize the yield of the appropriately assembled secreted antibody of the invention. Such modulation is performed, at least in part, by simultaneous translational modulation of the ribbons to the polypeptide components.

One technique for modulating translation strength is described in Simmons et al., US Patent 5,840,523. Variants of the Translation Beginning Region (TIR) are used in a citrus. For a given IRR, a series of amino acid sequence or variant nucleic acid may be created with a variation in translation forces, thus providing convenient means for adjusting this factor to the desired expression level of the specific chain. TIR variants can be generated from conventional demutagenesis techniques that result in codon changes that may alter the amino acid sequence, although silent changes in nucleotide sequence are preferred. IRR changes may include, for example, changes in the number or spacing of Shine-Dalgamo sequences, along with changes in the signal sequence. A preferred method for generating mutant signal sequences is the generation of a "codon bank" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (ie, silent changes). This can be accomplished by altering the third nucleotide position of each codon; In addition, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that may add complexity to the production of the bank. Mutagenesis dithomethod is described in detail in Yansura et al., (1992), METHODS: A Companion to Methods in Enzymol., 4: 151-158.

In one example, a set of vectors is generated with a TIR concentration range for each citrus present. This limited set provides a comparison of expression levels of each chain as well as the yield of full length products under various TIR concentration combinations. TIR concentrations can be determined by quantifying the level of expression of a gene reported as described in Simmons et al, US Patent 5,840,523. Based on the comparison of the translation strength, desired individual TI Rs are selected to be combined into expression vector constructs of the present invention.

Suitable prokaryotes for this purpose include marquebacteria and eubacteria, such as gram negative or gram positive organisms. Examples of useful bacteria include Escherichia (e.g., E.coli), bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (eg, Pseudomonas aeruginosa), Salmonella Typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla or Paracoccus. In one example, gram negative cells are used. In one example, E. coli cells are used as hosts for the invention. Examples include E. colicepa W3110 strains (Bachmann, Cellular and Molecular Bioloav, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; DepotATCC No. 27325) and its derivatives, including strains 33D3 having W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kanR genotypes (US Patent 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coliRV308 (ATCC 31608) are also suitable. These are preferably illustrative examples rather than limiters. Methods for constructing derivatives of any of the above-mentioned bacteria having the defined genotype are known in the art, for example, in Bas et al., Proteins, 8: 309-314 (1990). It is generally necessary to select suitable asbacteria taking into account the replicability of replicon in bacterial cells. For example, E. coli, Serratia or Salmonella species may be suitably used as hosts, when well-known plasmids such as plasmids pBR322, pBR325, pACYC177 or pKN410 are used for replicon construction.

Normally the host cell must secrete minimal amounts of proteolytic enzymes, and additionally protease inhibitors may be incorporated into the cell culture.

Antibody Production

Host cells are transformed with the expression vectors described above and cultured in conventional modified nutrient media as appropriate for promoter induction, selection of transformers or amplification of the genes encoding the desired sequences.

Transformation means introducing DNA into eukaryotic hosts in order for the DNA to be replicable, either as an extra-chromosome element or integrated into the chromosome. Depending on the host cell used, transformation is done using standard techniques suitable for such cells. Calcium treatment employing decalcium chloride is generally used for bacterial cells containing parecellular cells. Another transformation method employs polyethylene glycol / DMSO. Another technique still used is electroporation.

Prokaryotic cells used to produce the polypeptides according to the present invention are cultured in known art and suitable for culturing selected host cells. Examples of suitable media include lurium broth (LB) plus the necessary nutrient supplements. In preferred embodiments, the media also contain a selection agent, selected on the basis of the expression vector construct, to selectively permit growth of prokaryotic cells containing the expression vector. Ampicillin is added to the media, for example, for growth of cells expressing the ampicillin resistant gene.

Any necessary supplements other than sources of carbon, nitrogen and inorganic phosphate may also be included in appropriate concentrations introduced alone or as a mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at appropriate temperatures. For growth of E. coli, for example, the preferred temperature ranges from about 20 ° C to 39 ° C, more preferably from 25 ° C to 37 ° C, and about 30 ° C. The pH of the medium can be any pH from 5 to 9, depending mainly on the host organism. For E. coli, the pH is preferably about 6.8 to 7.4, preferably 7.0.

If an inducible promoter is used in the expression expression vector of the invention, protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the invention, PhoA promoters are used to control polypeptide transcription. Thus, cells are cultured in a phosphate limiting medium for induction. For example of the realization, the phosphate limiting medium is C.R.A.P. (see, for example, Simmons et al., J. Immunol. Methods. (2002), 263: 133-147). A variety of other inductors may be used according to the construction vector used, as is known in the prior art.

In one embodiment, the expressed polypeptides of the present invention are secreted and recovered from the host cell periplasma. Protein recovery typically involves disruption of the organism, usually by methods such as osmotic shock, sonication or lysis. After disruption of cells, cell debris or whole cells may be removed by centrifugation or filtration. Proteins may be further purified, for example, by affinity resin chromatography. Alternatively, the proteins may be transported to and isolated from the culture medium. The cells may be removed from the culture and the culture supernatant filtered and concentrated for further purification of the produced polypeptide. Expressed polypeptides may be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western Blot testing.

In one aspect of the invention, the antibody may be produced in large quantities by fermentation processes. Various large-scale batch feed fermentation procedures are available for the production of recombinant proteins. Large scale fermenters have a capacity of at least 1000 liters, preferably 1000 to 100,000 liters capacity. These fermenters use stirring impellers or other appropriate means for distributing oxygen and nutrients, especially glucose (a common source of carbon and energy). Small-scale fermentation generally refers to fermentation in fermenter which has a volumetric capacity of no more than about 100 liters and may range from about 1 liter to 100 liters.

In the fermentation process, induction of protein expression is typically initiated after cell growth under appropriate conditions to desired density, such as OD550 of about 180 to 220 at which the cells are at the beginning of the stationary phase. A series of inductors may be used according to the vector construct employed, as is known in the state of the art and described above. Cells may be cultured for shorter periods before induction. Cells are usually induced for about 12 to 50 hours, although longer or shorter induction time may be used.

To further increase the yield and quality of the polypeptide of the present invention, various defermentation conditions may be transformed. To improve assembly and secreted antibody, for example, additional vectors that overexpress chaperone proteins, such as Dsb (DsbA, DsbB, DsbC, DsbD and / or DsbG) or FkpA (peptidilprolil, trans-isomerase with chaperone activity) proteins can be used. used to co-transform host prokaryotic cells. Chaperone proteins have been shown to facilitate the formation of adequate hinge and solubility of heterologous proteins produced in bacterial host cells. Chen et al., J. Bio. Chem., 274: 19601-19605 (1999); US Patents 6,083,715 and 6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275: 17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem., 275: 17106-17113 (2000); Arie et al, Mol. Microbe !, 39: 199-210 (2001).

To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive) such as in prokaryotic host cells, certain proteolytic paraenzyme-deficient host lines may be used for the present invention. genetic mutation (s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, TonA, PhoA, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Some protease deficient E. coli strains are available and described, for example, in Joly et al, (1998) supra; Georgiou etal. US Patent 5,264,365, Georgiou et al. US Patent 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

In one embodiment, E. coli strains deficient in proteolytic enzymes transformed into one or more plasmids that overexpress chaperone proteins are used as host cells in the expression system of the invention.

Antibody Purification

In one example, the present produced antibody protein may be further purified to obtain substantially homogeneous preparations for further assays and uses. The following procedures are examples of suitable purification procedures: fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLG; chromatography on silica or cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE, ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75.

In one aspect, protein A immobilized by a solid phase is used for immunoaffinity purification of the antibody of the invention. Aprotein A is a 41 kD cell wall protein of Staphylococcus aureos that binds with high affinity to the Fc region of antibodies. Lindmark, et al (1983) J. Immunol. Meth 62: 1-13. The solid phase to which protein A is immobilized may be a column comprising a glass or silica surface, or a controlled porous glass column or acetic acid column. In some applications, the column is coated with a reagent, such as glycerol, possibly to prevent non-specific adhesion of contaminants.

As with the first purification step, the cell culture derivative preparation as described above can be applied to a solid phase immobilized protein A to allow binding of the specific antibody of interest to protein A. The solid phase would then be washed to remove contaminants not specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution. Antibody Production Using Eukaryotic Host Cells

Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an amplifier component, a promoter, and a transcription termination sequence.

(I) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide that has a specific N-terminal cleavage site of the mature protein or polypeptide of interest. The selected heterologous signal sequence is usually one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. In mammalian expression cells, mammalian signaling sequences as well as viral secretion leaders such as the herpes simplex gD signal are available.

The DNA for this precursor region is ligated in structure to the antibody encoding DNA. II) Replication Source Component

Generally, the origin of the replication component is not required for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the initial promoter.

(III) Selection Genetic Component

Cloning and expression vectors may contain a selection genus, also called a selection marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline; (b) complement autotrophic deficiencies; or (c) supplement key nutrients not available in the medium.

An example selection scheme utilizes a drug to arrest the growth of a host cell. Cells that are successfully transformed with a protein-producing heterologous gene confers drug resistance and thus survive the deletion regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selection markers for mammalian cells is those that allow identification of cells capable of absorbing antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-1 and II, preferably primate metallothionein, adenosine deaminase, ornithine decarboxylase genes. , and etc.

Cells transformed with the DHFR selection gene, for example, are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competing DHFR antagonist. An appropriate host cell when using wild-type DHFR is Chinese DHF-deficient ovarian (CHO) ovarian cell line (eg, ATCC CRL-9096).

Alternatively, host cells (particularly endogenous DHFR-containing wild-type hosts) transformed or co-transformed with DNA sequences encoding the wild-type DHFR antibody and other selectable marker such as aminoglycoside 3'-phosphotransferase (APH) may be selected by cell growth media in a medium containing a selectable marker selection agent such as aminoglycoside antibiotic, for example kanamycin, neomycin or G418. See US Patent 4,965,199.

(IV) Promoter Component

Cloning and expression vectors usually contain a promoter that is recognized by the host organism and is operably linked to the polypeptide nucleic acid (e.g., an antibody). Eukaryotic promoter consequences are known. Promoter sequences for eukaryotes are known. Virtually all genesis eukaryotes have an AT-rich region located about 25 to 30 bases upstream from the site of transcription initiation. Another sequence found at 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AAAAA sequence which may be the signal for addition of the poly-A tail at the 3' end of the coding sequence. All of these sequences are appropriately inserted into expression vectors for eukaryotes.

Transcription of polypeptides from vectors in mammalian host cells is controlled, for example, by promoters obtained from the viral genome, such as polyoma virus, chicken pox virus, adenovirus (such as Adenovirus 2), papilloma virus. bovine, avian disarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and preferably Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a deimmunoglobulin promoter, heat shock promoters, provided these drivers are compatible with host cell systems.

The initial and final promoter of the SV40 virus are conveniently obtained as SV40 restriction fragment which also contains the SV40 viral origin of replication. The human docitomegalovirus immediate initial promoter is conveniently obtained as a Hindlll E. restriction gene. A DNA expression system in mammalian hosts using the bovine papilloma virus as a vector is described in US Patent 4,119,446. A modification of this system is described in US Patent 4,601,978. See also Reyes et al, Nature 297: 598-601 (1982) on the expression of human interferon-β cDNA in mouse cells under the control of a herpes simplex virus promoter thymidine kinase. Rous's sarcoma can be used as a promoter.

(V) Component Amplifier Element

Transcription of an antibody polypeptide encoding DNA according to the present invention by higher eukaryotes is often increased by inserting an amplifying sequence into the vector. Many amplifier sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an amplifier of a eukaryotic cell virus will be used. Examples include the SV40 amplifier over the posterior side of the origin of replication (bp 100-270), the cytomegalovirus early promoter amplifier, the terminal side polyoma amplifier of the replication origin, and adenovirus amplifiers. See also Yaniv, Nature 297: 17-18 (1982) on promotion elements for activation of eukaryotic promoters. The amplifier may also be spliced into the vector at the 5'or 3 'position for the antibody coding sequence, but is preferably located at a 5' site of the promoter.

(VI) Transcription Termination Component

Expression vectors used in eukaryotic host cells will also contain sequences necessary for mRNA transcription termination and stabilization. Such sequences are usually available from the 5 'and occasionally 3' untranslated regions of the viral or eukaryotic DNA or cDNA. These regions contain transcribed denucleotide segments as polyadenylated fragments in the untranslated portion of the antibody-encoding mRNA. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector described therein. (VII) Selection And Transformation Of Host Cells

Suitable host cells for cloning or expression of DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of cultured vertebrate host cells (detained culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed macacotta kidney strain CV1 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in de-suspension culture, Graham et al., J. Gen. Virol. 36: 59 (1977)); hamster pup kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA. 77: 4216 (1980)); mouse sertolide cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey derim cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; human hepatoma lineage (Hep G2).

Host cells are transformed with the deconditioning or expression vectors described above for antibody production and cultured in conventional modified nutrient media as appropriate for promoter induction, selection of transformants, or amplifying the coding genes for the desired sequences.

(VIII) Cultivation Of Host Cells

Host cells used for antibody production according to the present invention may be cultured in a number of commercially available media, such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) media. and Dulbecco's EagleModified Medium ((DMEM), Sigma) which are suitable for culturing host cells. In addition, any medium described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Patents 4,767,704; US 4,657,866; US 4,927,762; US 4,560,655; or US5,122,469; WO 90/03430 and WO 87/00195 or US Patent Application 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides. (such as adenosine and thymidine), antibiotics (such as drugGENTAMICINA®), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, similar pH, are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

(IX) Antibody Purification

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space or secreted directly in the medium. If the antibody is produced intracellularly as a first step, the particulate fragments, whether host cells or lysed fragments, are removed, for example, by decentrifugation or ultrafiltration. When the antibody is secreted into the medium, supernatants from such expression systems are generally concentrated first using a commercially available protein concentration filter, for example an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis and antibiotics may be included to prevent the growth of unforeseen contaminants.

Antibody composition prepared from cells may be purified by using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of the affinity-binding Acomo ligand protein depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. The Apode Protein may be used to purify antibodies that are based on human γ1, y2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all human epara γ3 mouse isotypes (Guss et al, EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable arrays, such as controlled pore glass or poly (styrenedivinyl) benzene, allow higher flow rates and shorter processing times than can be achieved with agarose. When the antibody comprises a Ch3 domain, Bakerbond ABX ™ resin (J. T. Baker, Phillipsburg NJ, United States) is useful for purification. Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, Reverse Phase HPLC, silica chromatography, SEPHAROSE ™ heparin chromatography, chromatography on an anionic or cationic exchange resin (such as a polyaspartic acid column ), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step (s), the mixture comprising the antibody of interest and contaminants may be subjected to hydrophobic interaction chromatography at low pH using a time eluting at a pH of about 2.5 to 4.5. As an exemplary specific embodiment, preferably performed, low pH hydrophobic interaction chromatography is performed under low salt concentrations (such as about 0 to 0.25 M salt).

Activity Tests

The immunoglobulins of the present invention may be characterized by their physical / chemical properties and biological functions through various assays known in the art.

Purified antibodies may be further characterized by a number of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high performance liquid chromatography (HPLC), mass spectrometry, ion exchange, and papain digestion.

In specific embodiments, the immunoglobulins produced herein are analyzed for their biological activities. In some embodiments, the immunoglobulins of the present invention are tested for their antigen binding activity. Antigen binding assays that are known in the art and may be used herein include, but are not limited to, competitive or direct binding assays using techniques such as Western blots, radioimmunoassays, ELISA ("enzyme linked immunosorbent assay"), "type" immunoassays. sandwich ", immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.

Antibodies or antigen-binding fragments may still be selected for their functional activities, for example, agonist or antagonist activity. For example, anti-HER-2 antibodies may be selected by the ability to inhibit tyrosine phosphorylation in HER-2, inhibit cancer cell proliferation or induce cancer cells to apoptosis. Tests to identify and measure these activities are described, for example, in document W098 / 17797.

In addition, anti-DR5 antibodies may be selected for their ability to induce apoptosis in cancer cells and / or inhibit the function of inflammatory cells. In other embodiments, anti-DR5 antibodies are selected for their ability to compete with DR5 for binding to Apo-2L. In other embodiments, anti-human DR5 antibodies are selected by binding to murine DR5 and / or DR5 from cynomolgus monkeys. Assays for determining biological activity may be performed by methods known in the art, such as DNA fragmentation ( see, for example, Marsters et al., Curr. Biology, 6: 1669 (1996)), caspase inactivation, DR5 binding (see, for example, WO98 / 51793, published November 19, 1998. The level of apoptosis can be measured by identifying cytoplasm condensation, lost plasma membrane micro villi, nucleus segmentation, chromosomal DNA degradation or loss of mitochondrial function.This activity can be determined and measured, for example, by cell viability assays (such as Alamar blue assay or MTT assays), FACS analysis, caspase activation, DNA fragmentation (see, for example, Nicoletti et al., J. Immunol. Methods, 139: 2 71-279 (1991), poly-ADP ribose epimerimerase, PARP cleavage, and assays known in the art.

In one embodiment, such as the apoptosis assay involves performing twice the serial dilutions of the standard control and anti-DR5 antibody in 96-well culture plates. Apo-2 ligand (amino acids 114-281, described in PCT US 00/17579) is tested for breakdown effects. Colo-205 (20,000 cells-well) with human colon carcinoma (ATCC) cells are seeded in 96-well plates. The plates are incubated at 37 ° C for 24 hours. AlamazBlue (Jrek Diagnostic Systems, Inc.) is added to the wells within the last 3 to 24 hours of incubation time. Fluorescence is read using a 96-well plate fluorometer using the 530 nm excitation wavelength and 590 nm emission wavelength. Results are expressed as relative fluorescence units (RFU).

In one embodiment, the invention contemplates a modified antibody having some but not all effector functions, which makes this antibody a desirable candidate for many applications in which half-life of the antibody in vivo is important even though certain effector functions (such as complement and ADCC) are unnecessary or harmful. In certain embodiments, antibody Fc activities are measured to ensure that only the desired properties are maintained. An in vitro or in vivo decytotoxicity assay may be conducted to confirm reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody contains FcyR binding (and thus lacks ADCC activity) but retains the ability to bind to FcRn. Primary cells for ADCC mediation, NK cells, express FcyRIII only, while monocytes express FcyRI, FcyRII, and FcyRIII. The expression of FcR over hematopoietic cells is summarized in Table 3, p. 464, by Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). An example assay for determining the ADCC activity of a molecule of interest is described in US Patent 5,500,362 or US 5,821,337. Useful effector cells for these tests include peripheral blood mononuclear cells (PBMC) and Natural Killers (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be determined in vivo, as in an animal model such as that described in Clynes et al., PNAS (USA) 95: 652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. To determine complement activation, a CDC assay can be performed as described, for example, in Gazzano-Santoro et al., J Immunol.Methods, 202: 163 (1996). Determination of FcRn binding and in vivo clearance / half life can also be performed using methods known in the art, for example those described in the Examples section.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods of humanizing non-human antibodies are known in the art state. Preferably, a humanized antibody contains one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be essentially performed following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by means of replacing hypervariable deregion sequences with corresponding human antibody sequences.

Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent 4,816,567), wherein substantially less than one intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies, where some hypervariable deregion residues and possibly some FR residues are replaced by residues of analogous sites on rodent antibodies.

The selection of human variable domains, both light quantopes, to be used in the manufacture of humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequence of a rodent antibody is selected in comparison to any known human variable domain domain library. The human sequence that is closest to the rodent sequence is then accepted as the human structural region (FR) for the humanized antibody (Sims et al., (1993) J. Immunol., 151: 2296; Chothia et al., (1987) J. Mol. Biol., 196: 901). Another method utilizes a specific structural region derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same structure can be used for several different humanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623). It is additionally important that antibodies be humanized with high affinity retention for antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by means of a parental sequence analysis process and various conceptual humanized products using three-dimensional models of the parental and humanized sequence. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected immunoglobulinacandidate sequences. Inspection of these displays allows probable role analysis of residues in the functioning of the immunoglobulin-candidate sequence, that is, analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, the FR residues can be selected and combined from the imported receptor sequences so that the desired antibody characteristic such as greater affinity for the desired antigen (s) is achieved. Generally, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Variant Antibodies

In one aspect, the invention provides antibodies having modifications at the interface of Fc polypeptides comprising the Fc region, where modifications facilitate and / or promote heterodimerization. These changes include introducing a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, where the protuberance is positioned in the cavity to promote the complex between the first and second Fc polypeptide. Methods for producing antibodies with these modifications are known in the prior art, for example as described in US Patent 5,731,168.

In some examples, modification (s) are contemplated in the amino acid sequence (s) of the antibodies described herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Antibody amino acid sequence variants are prepared by introducing suitable nucleotides modifying the nucleic acid of the antibody, or by peptide synthesis. Such alterations include, for example, deletion, and / or insertion and / or substitution, of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion or replacement may be made to achieve the final construction, provided the final product has the desired characteristics. Amino acid changes can be introduced at the anti-point at which sequence is made.

To increase the half-life of the antibody or polypeptide containing the amino acid sequence of this invention, a recovered receptor recovery binding epitope may be incorporated into the antibody (especially an antibody fragment), as described, for example, in US Patent 5,739,277. For example, a nucleic acid molecule encoding a receptor salvage binding epitope can be ligated into a frame of a nucleic acid encoding a polypeptide sequence of the present invention such that the fusion protein expressed by the developed nucleic acid molecules contains the epitope. receptor binding linkage and a polypeptide sequence of the present invention. As used herein, the term "receptor recovery binding epitope" means an epitope of the Fc region of an IgG molecule (for example, IgGi, IgG2, IgG3 or IgG4) that is responsible for increasing the half-life in serum. of the IgG molecule. Alternatively or additionally, an increase or decrease in serum half life by altering the amino acid sequence of the Fc region of an antibody to generate altered FcRn binding variants. Antibodies with altered FcRn linkages and / or half-life changes are described in WO 00/42072 (Presta, L). WO 02/060919; Shields, R. L, et al., (2001) JBC 276 (9): 6591-6604; Hinton, P. R. (2004) JBC 279 (8): 6213-6216). In another embodiment example, serum half-life may also be increased, for example, by joining other polypeptide sequences. For example, antibodies of this invention or other polypeptide containing the amino acid sequences of this invention may be attached to albumin or a portion of albumin that binds to FcRn receptors or an albumin-binding peptide to bind to serum albumin, for example, such sequences. Polypeptides are disclosed in WO01 / 45746. In one example, the albumin peptide to be coupled comprises the amino acid sequence DICLPRWGCLW (SEQ ID No: 608). In another example, the half-life of a Fab according to this invention is increased by these methods. See also, Dennis, M. S., et al. (2002) JBC 277 (38): 35035-35043 for albumin-binding peptide sequences.

A useful method of identifying certain antagonist residues or regions that are preferred sites for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham eWells, Science, 244: 1081-1085 (1989). Here, a target residue or residue group is identified (e.g., charged residues such as arg, asp, his, lys and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction. dosamino acids with antigen. Those amino acid sites that demonstrate functional sensitivity to substitutions are then refined by introducing other variants or additional variants at the substitution sites or for these. Thus, although the site for introducing amino acid sequence variation is predetermined, the intrinsic nature of the mutation need not be predetermined. To analyze the performance of a mutation at a given site, for example, wing scan or random mutagenesis is conducted in the codon or target region and expressed antagonist variants are selected for the desired activity.

Amino acid sequence insertions include carboxy and / or amino-terminal fusions that range in length from one residue to polypeptides containing one hundred or more residues, as well as intra-sequential insertions of single or multiple amino acid residues. Examples of terminal insertions include a common N-terminal methionyl residue antagonist or antibody fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include the N- or C-terminal fusion of the antibody (e.g., for ADEPT) of an enzyme or polypeptide that increases the antagonist half-life in serum.

Another type of variant is a amino acid substitution variant. These variants contain at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of major interest for substitution mutagenesis include hypervariable regions, but changes in RF are also contemplated. Conservative substitutions are shown in Table 2 under the heading "preferred substitutions". If these substitutions result in changed biological activity, then more substantial changes, called "examples of substitutions" in the Table below, or as further described below with reference to amino acid classes can be introduced and products selected.

Table 2

<table> table see original document page 214 </column> </row> <table> Substantial modifications of the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect on the maintenance (a) of the main chain structure. polypeptide in the substitution area, for example in defoliation form or in helical conformation; (b) loading or hydrophobicity of the molecule at the desired site; or (c) the side chain volume. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)):

(1) Apolar: Wing (A), Go (V), Leu (L), Me (I), Pro (P), Phe (F), Trp

(W), Met (M)

(2) No polar charge: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) Acid: Asp (D), Glu (E)

(4) Basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues can be divided into groups based on common side chain properties:

(1) Hydrophilic: Norleucine, Met, Ala, Val, Leu, He;

(2) Neutral hydrophilics: Cys, Ser, Thr, Asn, Gln;

(3) Acid: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will cause a member of one of these classes to be replaced by another class. Such substituted residues may also be introduced at the conserved substitution sites or, more preferably, at the remaining (non-conserved) sites.

A particularly preferred type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., human and humanized antibody). Generally, the resulting variant (s) selected for further development will have modified (e.g., enhanced) biological properties with respect to the parent antibody from which it is generated. Briefly, several hypervariable site sites (eg, 6 to 7 sites) mutate to generate all possible amino substitutions at each site. Antibodies thus generated are displayed from filamentous phage particles fused to the M13 gene-encapsulated particle gene product. Phage-displayed variants are then selected according to their biological activity (e.g., binding affinity) as described in the present invention. In order to identify possible sites of hypervariable regions for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact waste and neighboring waste are candidates for replacement according to the techniques performed herein. Following generation of such variants, the variant frame is screened as described in the present invention and antibodies with superior properties in one or more relevant assays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a series of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and mutagenesis cassette. of a previously prepared variant or a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications into an Fc region of the antibody of the invention, thereby generating a variant Fc region. The human variant Fc region may comprise a sequence Fc region (e.g., IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions including that of a cysteine hinge .

According to this description and the teachings in the art, it is contemplated that, in some examples, an antibody of the invention may consist of one or more alterations compared to the wild type homologous antibody, for example, in the Fc region. These antibodies, however, retain substantially the same characteristics required for therapeutic autility compared to their wild-type counterpart. For example, some changes may be made in the Fc region that alter (ie, improve or decrease) the binding to C1q. and / or complement-dependent cytotoxicity (CDC), for example, as described in document WO99 / 51642. See also Duncan and Winter, Nature 322: 738-40 (1988); US Patent 5,648,260; US patent. 5,624,821, and WO94 / 29351 relating to other examples of variant Fc region.

IMMUNIZED

The present invention also relates to drug conjugated immunoconjugates (AADs), comprising an anti-conjugate to a cytotoxic agent such as a chemotherapeutic agent, drug, growth inhibitory agent, toxin (e.g., a bacterially active, fungal toxin toxin). , plant or animal, or fragments thereof) or a radioactive isotope (ie, a radioconjugate). Using drug-conjugated antibodies for local delivery of cytotoxic or cytostatic agents, ie drugs to kill or inhibit cellulastumor in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26: 151-172; US Patent 4,975,278) theoretically allow drug delivery to the target cell of part of the drug. intracellular accumulation, where systemic administration of these unconjugated agents may result in unacceptable levels of toxicity in normal cells as tumor cells that need to be eliminated (Baldwin et al. (1986) Lancetpp. (March 15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review On," Monoclonal Antibodies'84: Biological! And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506) .Then the maximum efficacy is sought with minimum toxicity. Both polyclonal antibodies and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al. (1986) Supra). Astoxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small toxin molecules such as geldanamycin Mandler et al (2000) Jour. ofthe Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13: 786-791), maytansinoids (EP Patent 1391213; Liu et al. (1996) Proc. Natl. Acad.Sei. USA 93: 8618-8623), and caliceamycin (Lode et al (1998) Cancer Res.58 : 2928; Hinman et al (1993) Cancer Res. 53: 3336-3342). Toxins can realize their cytotoxic and cytostatic effects by mechanisms including tubulin alleviation, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to antibody or receptor protein ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen / ldec) is an antibody-radioisotope conjugate composed of a lgG1 kappamurino monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant W11-linked quelantel-bound In111 or Y90 radioisotope et al (2000) Eur. Jour. Nucl. Med. 27 (7): 766-77; Wiseman et al (2002) Blood 99 (12): 4336-42; Witzig et al (2002) J. Clin. 20 (10): 2453-63, Witzig et al (2002) J. Clin Oncol 20 (15): 3262-69). Although ZEVALIN has activity against non-Hodgkin B-cell lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARG ™ (gemtuzumab ozogamycin, Wyeth Pharmaceuticals), a drug-conjugated antibody composed of an acaliceamicin-linked hu CD33 antibody, was approved in 2000 for the treatment of injection myeloid leukemia (Drugs of the Future (2000) 25 (7): 686; US Patent 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US5767285; US 5773001). Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate composed of the huC242 antibody bound via SPP, the maytansinoid fraction of the drug, bisulfide DM1, is advancing in phase II trials for the treatment of CanAg-expressing cancers, for example, pancreatic , gastric among others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate composed of the anti-prostatic membrane antigen (PSMA) -specific monoclonal antibody linked to the maytansinoid fraction of the drug, DM1, is under development for treatment. potential of deprostate tumors. Auristatin, auristatin E (AE) and ammonomethyluristatin (MMAE) peptides, synthetic dolastatin analogues, conjugated to chimeric monoclonal antibodies cBR96 (specific for Lewis Y carcinoma) and cAC10 (specific for CD30 in haematological malignancies) (Doronina et al ( 2003) Nature Biotechnology 21 (7) 778-784) and are under therapeutic development.

Chemotherapeutic agents useful in the generation of such immunoconjugates are described above herein. Enzyme-reactive toxins and fragments thereof which may be used include diphtheria A chain, diphtheria toxin non-binding active fragments, exotoxin A chain (dePseudomonas aeruginosa), castor A chain, abrin A chain, demodecin A chain, alpha-sarcin , Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordicacharantia inhibitor, curcin, crotin, Sapaonaría officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionucleotides are available for the production of radioconjugate antibodies. Examples include Bi212, 131, In131, Y90 and Re186. Antagonist and cytotoxic agent conjugates can be manufactured using a series of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylditiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis- (para-azidobenzoyl) -hexanediamine), bis-diazonium derivatives (such as bis- (para- diazoniobenzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminopentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antagonist. See document WO 94/11026.

Conjugates of an antagonist and one or more small demolecular toxins, such as calicheamicin, maytansinoids, trichothene and CC1065 and derivatives thereof which also exhibit toxin activities, are contemplated herein.

Maytansine and Maytansinoids

In some exemplary embodiments, the immunoconjugate comprises the antibody of the invention conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from Maytenus serrata (US Patent 3,896,111). It was then found that certain microbes also produce maytansinoids, such as maytansinol and maytansinol C-3 esters (US Patent 4,151,042). Synthetic maytansinols, their derivatives and analogs are described, for example, in US Patents 4,137,230, US 4,248,870, US 4,256,746, US 4,260,608, US 4,265,814, US 4,294,757, US 4,307,016, US 4,308. 268, US 4,308,269, US 4,309,428, US 4,313,946, US 4,315,929, US 4,317,821, US 4,322,348, US 4,331,598, US4,361,650, US 4,364,866, US 4,424,219, US 4,450,254, US 4,362,663 and US 4,371,533, the disclosures of which are expressly incorporated herein by reference.

Maytansinoid Antibody Conjugates

In an attempt to improve its therapeutic index, maytansinae and maytansinoids have been conjugated to antibodies that specifically bind to tumor cell antigens, maytansinoid-containing immunoconjugates and their therapeutic use are disclosed, for example, in US Patent 5,208,020, US 5,416,064 and European Patent EP 0 425 235 B1, disclosures of which are expressly incorporated herein by reference. Liu et al., Proc. Natl. Acad. Know. USA 93: 8618-8623 (1996) has described immunoconjugates comprising a maytansinoid designated DM1 bound to the human cancer-monoclonal C242 monoclonal antibody. The conjugate has been shown to be highly cytotoxic to cultured colon cancer cells, and has shown anti-tumor activity in an in vivo tumor growth analysis. Chari et al., Cancer Research 52: 127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linkage to the murine A7 antibody ligand to a human colon cancer cell line antigen, or other murine TA monoclonal antibody. .I binding to the WSR.-2lneu oncogene. The cytotoxicity of the TA.I-maytansinoid conjugate was tested in vitro on SK-BR-3 human breast cancer cell alignment, which expresses 3 x 10 5 HER-2 surface antigens. per cell. The conjugated drug achieves a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by the increase in the number of maytansinoid molecules per antibody molecule. A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Maytansinoid Antibody Conjugates (Immunoconjugates)

Antibody-maytansinoid conjugates are prepared chemically by binding an antibody to a maytansinoid molecule without significantly decreasing the biological activity of the antibody or the maytansinoid molecule. An average of 3-4 antibody molecule-conjugated maytansinoid molecules showed efficacy in amplifying cytotoxicity in target cells without negatively affecting antibody function or solubility, although even one antibody / toxin molecule was expected to amplify acitotoxicity upon use of an antibody. naked. Maytansinoid are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are described, for example, in US 5,208,020 and other non-patent publications referred to hereinbelow. Preferred maytansinoids are maytansinol and maytansinol analogs modified in the annular aromatic or other positions of the maytansinol molecule, such as various maytansinol esters.

There are several art-known linker groups for the manufacture of antibody and maytansinoid conjugates, including, for example, those described in US Patent 5,208,020 or EP0,425,235 B1 and Chari et al., Cancer Research 52: 127. -131 (1992). Linking groups include disulfide groups, thioether groups, labile acid groups, photolabile groups, peptidase labile groups or esterase labile groups as described in the patents identified above.

Antibody and maytansinoid conjugates can be manufactured using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylditio) propionate (SPDP), succinimidyl-4- (N-cyclohexane-1-carboxylate) -maleimidomethyl), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis azide compounds (such as bis (para-azidobenzoyl) ) -hexanediamine), bis-diazonium derivatives (such as bis- (para-diazoniobenzoyl) ethylenediamine), diisocyanates (such as 2,6-diisocyanate detoluene) and bis-active fluorine compounds (such as 1,5-difluoro -2,4-dinitrobenzene). In certain embodiments, coupling agents include N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978)) and N-succinimidyl propionate. succinimidyl-4- (2-pyridylthio) (SPP) to provide disulfide bonding.

The binder may be attached to the maytansinoid molecule in several positions, depending on the type of binding. An ester bond may be formed, for example, by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at position C-3 which has a hydroxyl group, at position C-14 modified with hydroxymethyl, at position C-15 modified with a hydroxyl group and at position C-20 which has a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

Calicheamicin

Another immunoconjugate of interest comprises an anti-conjugate to one or more calicheamicin molecules. The antibiotic family calicheamicin is capable of producing phytadupla DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001 and US 5,877,296. (all from the American Cyanamid Company). Decaliqueamycin structural analogs that may be used include, but are not limited to, γ-i ', Î ±, Î ± 3', N-acetyl-γ1, PSAG and 2β (Hinman et al., Cancer Research 53: 3336-3342 ( 1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and AmericanCyanamid US patents cited above). Another antitumor drug to which the antibody can be conjugated is QFA, which is an antifolate. Both calicheamicin and FFA contain intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody-mediated internalization greatly increases their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that may be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the LL-E33288 complex family of known agents collectively described in US 5,053,394 and US 5,770,710, as well as speramycin ( US Patent 5,877,296).

Enzymatically active toxins and fragments thereof which may be used include diphtheria A chain, diphtheria datoxin non-linker active fragments, exotoxin A (Pseudomonas aeruginosa) chain, castor A chain, abrin A chain, modeccin A chain, alpha-sarcin , Allurite fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, inhibitor of Saaponaria officinalis, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, WO 93/21232, published October 28, 1993.

The present invention further contemplates an antagonist conjugated to a compound with nucleolytic activity (e.g. ribonuclease or a DNA endonuclease such as deoxyribonuclease; DNase).

For selective tumor destruction, the antibody may contain a highly radioactive atom. A number of radioactive isotopes are available for the production of radioconjugate antagonists. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a despin tag for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radiolabeling or the like may be incorporated into the conjugate in known ways. The peptide may be, for example, biosynthesized or may be synthesized by chemical amino acid synthesis using appropriate amino acid precursors involving, for example, hydrogen fluorine. Marks such as tc99m, I123, Re186, Re188 and In111 may be linked via a cysteine residue in the peptide. Yttrium-90 can be bound through a lysine residue. The lodogen method (Fraker et al. (1978), Biochem. Biophys. Res. Commun. 80: 49-57) can be used for the incorporation of iodine-123. Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press, 1989) describes other methods in detail.

Antibody and cytotoxic agent conjugates can be manufactured by a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylditio) propionate (SPDP), succinimidyl-4- (N-cyclohexane-1-carboxylate) -maleimidomethyl), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCI), active esters (such as di-succinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis- (for -azidobenzoyl) -hexanediamine), bis-diazonium derivatives (such as bis- (para-diazoniobenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bisactive fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitettaet al., Science 238: 1098 (1987). 14-labeled carbon 1-isothiocyanatobenzyl-3-methyldiethylenetriaminopentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucletide to the antibody. See document WO 94/11026. The binder may be a "cleavable binder" that facilitates the release of the cytotoxic drug into the cell. For example, a labile acid binder, peptidase-sensitive binder, photolabile binder, dimethyl binder or disulfide-containing binder (Chari et al., Cancer Research 52: 127-131 (1992); US Patent 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are not limited to, ADC prepared with cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) which are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL, USA). See pages 467-498 of Applications Handbook andCatalog 2003-2004.

Preparation of Drug-Conjugated Antibodies

In the drug antibody (ADC) conjugates of the invention, an antibody conjugated (Ab) to one or more drug fractions (D), for example, about 1 to 20 drug fractions per antibody, via a ligand (L) . The OADC of formula I may be prepared by a variety of protocols employing organic chemistry reactions, circumstances and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of an antibody with a bivalent linker to form Ab-L via of a covalent bond, followed by reaction with the drug fraction D; and (2) reacting a nucleophilic group of a drug moiety with a divalent ligand reagent to form D-L by covalent bonding followed by nucleophilic group dyeing of an antibody.

Ab- (L-D) p

Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, for example lysine, (iii) side chain thiol groups, for example cysteine, and (iv) hydroxyl sugar or amino groups where the antibody is glycosylated. The amino, thiol and hydroxyl groups are nucleophilic and capable of reacting to covalently bonds with the electrophilic groups in fractions of ligands and ligands including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) Benzyl alkyls and halides such as haloacetamides; (iii) groups of aldehydes, ketones, carboxyl emaleimide. Certain antibodies have reduced interchain bonds, i.e. cysteine bridges. Antibodies can be made reactive by conjugation with the binding reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive nucleophilic thiols. Additional nucleophilic groups may be introduced into antibodies by reacting the lysines with 2-iminothiolane (Traut's reagent) resulting in the conversion of an amine to a thiol.

Antibody conjugates with drugs of the invention may also be produced by modifying the antibody to introduce the electrophilic region, which may react with nucleophilic substituents on the reagent or drug. Glycosylated antibody sugars may be oxidized, for example, with periodate oxidation reagents to form aldehyde or ketone groups which may react with the reagent-binding amino group or drug moieties. The resulting imine groups of Schiff may form a stable bond, or may be reduced, for example by the borohydride reagent to form stable amino bonds. In one embodiment, the reaction of the carbohydrate moiety of an anti-polyglycosylate with galactose oxidase or sodium metaperiodate may form carbonyl groups (aldehyde and ketone) in the protein which may thus react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues may react with sodium meta-periodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). Talaldehyde may react with a reagent binder or a fraction of droganucleophilic.

Similarly, nucleophilic groups in a drug moiety include, but are not limited to: amino, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide groups capable of reacting as covalent bonds with the electrophilic groups. in binder and binder reagent fractions including: (i) esters, such as NHS esters, HOBt esters, haloformates and halidesacids; (ii) Benzyl alkyls and halides such as haloacetamides; (iii) dosaldehyde, ketone, carboxyl and maleimide groups.

Alternatively, a fusion protein may be made comprising the antibody and cytotoxic agent, for example by recombinant techniques or peptide synthesis. The length of the DNA may comprise the respective regions encoding the two conjugated portions are adjacent or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

However in another embodiment, the antibody may be coupled to a "receptor" (such as streptoavidin) for tumor autilization by pre-directing the antibody-receptor conjugate that is administered to the patient, followed by removal of the conjugate that is not selected to the tumor. receptor and is in circulation by the use of a cleaning agent, and then a "binder" (e.g., avidin) is administered in which it is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Derivative Antibodies

Antibodies of the invention may be further modified to contain additional non-proteinaceous molecules which are known in the art and readily available. In one example, molecules suitable for derivatization of the antibody are water soluble polymers.

Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, pyrrolidone polychloride, poly-1,3-dioxolane, poly-1,3, 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyethylene oxide polypropylene oxide copolymers, polyalcoholes (e.g. glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycolpropionaldehyde may have advantages in the manufacturing process due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of antibody-coupled polymers may vary, and if more than one polymer is attached, the polymers may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations, including but not limited to, specific properties or functions of the antibody to be improved, whether the derived antibodies will be used within therapy, etc. .

Pharmaceutical Formulations

Therapeutic formulations comprising the inventive antibody are prepared are prepared for storage by admixing an antibody of the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). )), in the form of lyophilised formulations, aqueous solutions or other dry formulations. Acceptable carriers, excipients or stabilizers are non-toxic to patients at the dosages and concentrations employed and include buffers such as phosphate, citrate, histidine and other organic acids, antioxidants including ascorbic acid and methionine; preservatives (such as deoctadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenolic, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol emeta-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn protein complexes); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

The formulation of the present invention may also contain more than one active compound as required for the specific indication to be treated, preferably said compounds exhibit complementary activities that do not harm each other. Such molecules are suitably presented in combination in amounts that are effective for the purpose intended.

The active ingredients may also be stored in microcapsules prepared, for example, by preservation or interfacial polymerization techniques, such as hydroxymethylcellulose microcapsules or gelatin and poly (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. , liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Ed. Osol, A. (1980).

The formulations to be used for in vivo administration should be sterile. For example, by filtration through sterile filtration membranes.

Extended release preparations may be manufactured. Suitable examples of extended release preparations include semipermeable arrays of solid hydrophobic polymers containing the inventive globulin, which are in the form of molded articles such as films or microcapsules. Examples of extended release matrices include polyesters, hydrogels (such as poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), poly-lactides (US Patent 3,773,919), L-glutamic acid and L- ethyl glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid and glycolic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid glycolic acid and leuprolide acetate) and acid poly (D) - (-) - 3 -hydroxybutyric. Although polymers such as ethylene vinylacetate and lactic acid glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a longer period of time, they may denature or aggregate as a result. exposure to moisture at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be planned for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be the intermolecule SS deletion formation through thiosulphide interchain, stabilization can be achieved by sulfhydryl residues, lyophilization of acidic solutions, moisture content control using appropriate additives, and development of polymer matrix decompositions. specific.

Use

An antibody of the invention may be used, for example, in vivo, ex vivo and in vivo therapeutic methods. Antibodies of the invention may be used as an antagonist to partially or totally block antigen specific activity in vitro, ex vivo and / or in vivo. In addition, even some of the antibodies of the invention may counteract the antigen activity of other species. Thus, the antibodies of the invention may be used to inhibit the activity of a specific antigen, for example in a cell culture containing the antigens, in humans or other mammals, in individuals who have the antigen with which an invention antibody cross-reacts (eg. chimpanzee, baboon, tamarin, cinomolgus and Rhesus, pig or mouse). In one example, the antibody of the invention may be used to inhibit antigen activity by exposing the antibody to the antigen in such a way that antigen activity is inhibited. In one example, the antigen is a human protein molecule.

In one embodiment, an antibody of the invention may be used in a method for inhibiting an antigen in a patient suffering from a disease in which antigen activity is detrimental, comprising administering to the patient an antibody of the invention in which the patient's antigen activity is inhibited. In one example, the antigen is a human protein molecule and the patient is a human patient.

Alternatively, the patient may be a mammal expressing the antigen to which the antibody of the invention binds. Additionally, the patient may be a mammal into which the antigen has been introduced (for example, antigen administration or expression of a transgenic antigen). The antibody of the invention may be administered to a human patient for therapeutic purposes. In addition, the antibody of the invention may be administered to a non-human mammal expressing the antigen with which the antibody cross-reacts (e.g., a primate, pig or rat) for veterinary purposes or as an animal model of human disease. With respect to the latter, such animal models may be useful for assessing the therapeutic efficacy of the antibodies of the invention (e.g., finger testing and time course of administration).

Antagonist or blocking antibodies of the invention which are useful include, for example, but not limited to, anti-HER-2 antibodies. The anti-HER2 antibody of the invention may, for example, be used to treat, inhibit, slow progression, prevent / delay recurrence, alleviate or prevent diseases, conditions or conditions associated with abnormal expression or activity of one or more antigen molecules, including but not limited to malignant and benign tumors; lymphoid and non-leukemic malignancies; neuronal, glial, astrocital, hypothalamic, and other glandular, macrophage, epithelial, stromal, and blastocellic disorders; inflammatory and immune related edisturbances.

In one aspect, blocking antibodies of the invention are specific for a ligand antigen, and inhibit antigen activity by blocking or interfering with ligand-receptor interaction involving the ligand antigen, thereby inhibiting the corresponding cellular and molecular signaling pathway or other events. The invention also has the advantage of having specific antibody receptors that do not necessarily prevent ligand binding but interfere with receptor activation, thereby inhibiting any responses that would normally be initiated by ligand binding. In certain embodiments, the invention also encompasses antibodies that preferentially or exclusively bind to the receptor-ligand complex. An antibody of the invention may also act as an agonist of a specific receptor antigen, thereby potentiating, enhancing or partially or fully activating the activities of the ligand mediated by receptor activation.

Disorders or conditions and diagnostic tests associated with HER-2 are described in US Patent 6,387,371, which is incorporated herein by reference. See also document W098 / 17797. Administration to a patient of a therapeutically effective amount of anti-HER-2 receptor antibodies inhibits tumor cell growth and is useful in treating cancer. Trastuzumab (Genentech, Inc.) is a recombinant human monoclonal antibody directed against the HER-2 extracellular domain for the treatment of cancers that overexpress HER-2 / has the HER-2 gene amplified, especially in metastatic breast cancer (MBC). These anti-antibodies are useful in treating other cancers especially those expressing HER-2. The antibody may also be administered to patients in combination with other therapies such as paclitaxel or Tarceva®.

In some embodiments, an anti-DR5 antibody induces apoptosis in cancer cells. In some embodiments, the anti-DR5 antibody is a DR5 agonist. In other embodiments, the antibody competes with DR5 for Apo-2L binding.

As noted above, DR5 antibodies of the invention have various uses. For example, agonistic DR5 antibodies may be employed in methods for treating mammalian conditions such as cancer or immunity-related diseases. Immune-related conditions include rheumatoid arthritis, systemic lupus erythematosus, scleroderma, idiopathic inflammatory myopathies, sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, thyroiditis, immunorelated kidney disease, such as glomerulonephritis, autoimmune disease, demyelinating disease, autoimmune disease such as psoriasis, inflammatory infiltrated lung diseases, and allergic diseases such as asthma.

Diagnosis of the various pathological conditions in mammals described herein may be made by the qualified physician. Diagnostic techniques are available in the prior art which allow, for example, the diagnosis or detection of cancer or immune related diseases in a mammal. For example, cancers may be identified through techniques, including but not limited to, palpation, blood analysis, X-ray, NMR, and the like. Immune-related diseases can also be easily identified. For example, in systemic lupus erythematosus, the central mediator of the disease is the production of the patient's own aprotein / tissue reactive autoantibodies and the subsequent generation of immune-mediated inflammation. Multiple organs and systems are clinically affected including kidney, lung, musculoskeletal system, mucocutaneous, eyes, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood. Doctors are familiar with a number of diseases in which immune and / or inflammatory response intervention have benefited.

In a specific embodiment an immunoconjugate comprising an antibody with a cytotoxic agent is administered to the patient. In some embodiments, the immunoconjugate and / or antigen to which it is bound is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in cell death to which it binds. In a preferred embodiment, the cytotoxic agent combines or interferes with nucleic acid in cancer cells. Examples of cytotoxic ditosagents including chemotherapeutic agents cited herein (e.g., matasinoids or calichiamycin), ouribonucleases radioactive isotypes and DNA endonucleases.

Antibodies of the invention may be used alone or in combination with other compositions in a therapy. For example, an antibody of the invention may be co-administered with another antibody, chemotherapeutic agent (s) (including cocktails of chemotherapeutic agents), other cytotoxic agent (s), anti-angiogenic agent (s). ), cytokines or growth inhibitory agent (s). In which the antibody of the invention inhibits the growth of a tumor, which may, if desired, be combined with another additional therapeutic agent which also inhibits tumor growth. For example, an antibody of the invention may be combined with an anti-VEGF antibody (e.g., AVASTIN) and / or anti-ErbB antibodies (e.g., anti-HER-2 antibodies, HERCEPTIN®), in a treatment regimen, e.g. in the treatment of one of the diseases described herein, including colorectal cancer, metastatic breast cancer and kidney cancer. Alternatively, or in addition, the patient may receive combined radiotherapy (eg, external beam irradiation or therapy with a labeled agent such as an antibody). ). Such combined therapies referred to above include combined administration (in which two or more agents are included therein, or in separate formulations), and separate administration, in which case administration of the antibody of the invention may occur prior to and / or after administration of the therapy. or adjuvant therapies.

The antibody of the invention (and adjunct therapeutic agent) is / are administered by any appropriate means including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the antagonist is suitably administered by pulse infusion, particularly with decreasing doses of the antagonist. Preferably, the dosage is provided by injections, most preferably intravenous or subcutaneous injections, depending on whether the administration is rapid or chronic.

The antibody composition should be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular dysfunction to be treated, the particular mammal to be treated, the clinical condition of the patient, the cause of the dysfunction, the place of delivery of the agent, the method of administration, the schedule of administration and other factors known to physicians. . The antibody need not be, but is optionally formulated with one or more agents commonly used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of the anti-powder present in the formulation, the type of dysfunction or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described above or about 1 to 99% of the dosages described herein. For the prevention or treatment of disease, the appropriate dosage of antibody (when used alone or in combination with other agents such as chemotherapy) will depend on the type of disease being treated, the type of antibody, the severity and the course of the disease, whether the antibody is administered for external therapeutic preventive purposes, prior therapy, the patient's medical history, antibody reaction, and the attending physician's discretion. The antibody is suitably administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 µg / kg to 15 mg / kg (such as 0.1 mg / kg to 10 mg / kg) of antibody is a possible starting dose for administration to the patient, for example. , by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 µg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is continued until desired suppression of disease symptoms occurs. An exemplary antibody dosage will be in the range of about 0.05 mg / kg to 10 mg / kg.

Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, as weekly or every three weeks (for example, such that the patient receives about two to about twenty, such as about six doses of the antibody). Higher initial dose of attack, followed by one or more lower doses, may be administered. Example dosing regimen comprises administration of an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the antibody. However, other dosage regimens may be helpful. The extension of this therapy is easily monitored by conventional techniques and tests.

Manufactured Articles

In another aspect of the invention, a manufactured article containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above are provided. The manufactured article consists of a container and label or a package insert on or associated with the container. Suitable containers include, for example, vials, syringes, blisters, etc. The containers may be made of a variety of materials such as glass or plastic. The container holds a composition which is alone or is combined with another composition effective to treat and prevent and / or diagnose the condition and which may have a sterile access port (for example, the container may be an intravenous solution pouch or a vial having a controller). (pierceable stopper) placed through a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is useful for the treatment of choice. Additionally, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention, and (b) a second container with a composition contained therein comprising a cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package leaflet indicating which compositions may be used to treat a particular case. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as water. bacteriostatic injection (BWFI), phosphate buffered saline, Ringer's solution and Other desirable materials may also be included from a commercial and usage perspective, including other buffers, diluents, filters, needles and syringes.

After the invention has been described generally, it will be more readily understood by reference to the following examples which are provided for purposes of illustration and are not intended to limit the invention.

Examples

Example I

Building Fab Libraries Displayed By Phage With CDR Waste Enriched In Tyr, Ser.Gly And Arg

Phage-displayed Fab libraries were constructed using a phagemid vector, Fab-C, which resulted in the display of divalent Fab bivalent dimers by a free cysteine inserted between the heavy chain Fab and the C-terminal domain of the capsid secondary protein (P3C) gene-3 ). This vector was constructed as described in US Patent Application 2005/0119455 and in Lee et al., J. Immunol. Meth.284: 1119-132 (2004). The vector (schematically illustrated in Figure 5) includes the variable domains of the humanized 4D5 antibody under the Ptac IPTG-inducible promoter control. Humanized 4D5 antibody has the human consensus sequence of the structural region of light and heavy chains, and CDR regions of a HER-2-specific monoclonal antibody. Methods for the production of anti-HER-2 antibodies and variable domain sequence identity are provided in US Patent 5,821,337 and US 6,054,297.

Four libraries were built: YSGR-A, YSGR-B, YSGR-C, and YSGR-D. Libraries were constructed with randomized residues on all three CDR3 heavy and light chain CDRs. Each library was reacted at CDRL3 positions 91-94 and 96, CDRH1 positions 28, 30.52-54, 56 and 58, and at positions 95, 96, 97, 98, 99, 100, 100a, 100b and 100c of CDRH3. The type and ratio of amino acids allowed for each randomized deposition are illustrated in Figure 8. In addition, the length of the CDRH3 was varied using oligonucleotides that replaced wild type setecodons at positions 95 to 100a with six to seventeen codons. Thus, in certain cases, the codon corresponding to position 100a of the heavy chain was not present (for example, when mutagenesis was performed with mutagenic oligonucleotides H3-A6 (SEQ ID NO: 35), H3-B6 (SEQ ID No: 47) , H3-C6 (SEQ ID No: 59) or H3-D6 (SEQ ID No: 71), as described below: See Figure 9A-DO Type and the amino acid ratio allowed at these positions were the same as those described in Figure 8 for the positions. 95-100a of CDRH3.

Libraries were constructed using the Kunkel method (Kunkel, TA, Roberts, JD & Zakour, RA, Methods Enzymol. (1987), 154,367-382), with previously described methods (Sidhu, SS, Lowman, HB, Cunningham, BC & Wells, JA, Methods Enzymol. (2000), 328, 333-363).

A single "stop template" version of the Fab display vector, Fab-C, was used to generate all four libraries, as described in Example 1.

Mutagenic oligonucleotides with degenerating codons in the positions to be diversified were used to simultaneously (a) introduce diversity into CDR and (b) repair stop codons. The sequences of these mutagenic oligonucleotides are shown in Figures 9A-9D. For all libraries, diversity was introduced into CDR-H1, CDR-H2 and CDR-H3 with oligonucleotides H1, H2e and L3, respectively (SEQ ID Nos. :). For library YSGR-A, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-A6, H3-A7, H3-A8, H3-A9, H3-A10, H3-A12, H3-A12, H3-A13 , H3-A14, H3-A15, H3-A16, and H3-A17 (SEQ ID Nos: 35-46). For the YSGR-B library, a diversity of CDR-H3 with an equimolar mixture of H3-B6, H3-B7, H3-B8, H3-B9, H3-B10, H3-B12, H3-B oligonucleotides was introduced. B13, H3-B14, H3-B15, H3-B16, and H3-B17 (SEQID Nos: 47-58). For the YSGR-C library, a CDR-H3 diversity was introduced with an equimolar mixture of oligonucleotides H3-C6, H3-01, H3-C8, H3-C10, H3-C10, H3-C12, H3- C13, H3 -C14, H3 -C15, H3 -C16, and H3 -C17 (SEQ ID Nos: 59-70). For the YSGR-D library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-D6, H3-D7, H3-D8, H3-D9, H3-D10, H3-D12, H3-D13 equimolar mixture. , H3-D14, H3-D15, H3-D16, and H3-D17 (SEQ ID Nos: 71-82). Each of the H3-A6 to H3-A17 mutagenic oligonucleotides (SEQ ID NOS: 35-46), H3-B6 to H3-B17 (SEQ ID NOS: 47-58), H3-C6 to H3-C17 (SEQ IDNos: 59 -70) and H3-D6 to H3-D17 (SEQ ID Nos: 71-82) encoded an alanine at position 93 of the heavy chain. Mutagenic oligonucleotides for all CDRs were randomized to be incorporated simultaneously into a single mutagenesis reaction, thus the simultaneous incorporation of all mutagenic oligonucleotides resulted in the introduction of the projected diversity at each position and simultaneously repairing all TAA stop codons. Thus, an open reading frame was generated to encode a Fab library fused to a homodimerizing cysteine bridge and P3C. Following mutagenesis, the four libraries were combined to create a single library called the YSGR-AD library.

Mutagenesis reactions were electroporated into E. coli SS320 (Sidhu et al., Supra). Transformed cells were grown overnight in the presence of M13-K07 phage helper (New England Biolabs, Beverly, MA) to produce phage particles that encapsulate phagemid DNA and display Fab fragments on their surfaces. The combined library contains more than 3x10 unique members.

EXEMPLQ2

Selecting Specific Antibodies from YSGR-ADNaives Libraries

YSGR-AD library phage (described in Example 1, above) were cyclized by ligation selection steps to enrich human DR5 or HER-2 ligand clones. Selections were made using binding methods described above (Sidhu et al, Supra).

A DR5 sequence is shown in Table 1. An extracellular DR5 domain as shown in Table 1 is used in ligation selection. Similarly, a sequence of the extracellular domain of human HER-2 is prepared as described by Franklin MC. Carey KD. Vajdos FF.Leahy DJ. from Vos AM. Sliwkowski MX, Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex, Cancer Cell. 5 (4): 317-28, 2004. Consequence for HER-2 ECD (amino acids 23-646) is provided in ProteinDataBank Record 1S78 (2004).

Maxisorp Immunoplates 96-well NUNC plates were coated overnight at 4 ° C with 5 µg / ml target protein (human DR5 or human HER-2) and blocked for 2 hours with a PBT (additionally 0.2% BSA and phosphate buffer) solution. 0.05% Tween 20 (Sigma)).

Following overnight growth at 37 ° C, the phages were concentrated by precipitation with PEG / NaCl and resuspended in PBT as described previously (Sidhu et al, Supra). Phage solution (about 1012 phage / mL) was added to the coated Immunoplate plate. After two hours of incubation to allow phage to bind, the plates were washed ten times with PBT. Bound phage were eluted with 0.1 M HCl for ten minutes and the eluents were neutralized with 1.0 M Tris base. The eluted phages were amplified in E.coliXL-1-blue and used for further selection steps. Libraries were subjected to five steps of target protein contraction selection. Individual clones from each selection step were cultured in 96-well wells in 500 µl of 2YT broth supplemented with carbenicillin and M13-K07. The culture supernatant was used directly on ELISA phage (Sidhu et al., Supra) to detect phage-displayed Fabs that bound on the target protein coated plates but did not seal the BSA coated plates. Specific ligands were defined as those phage clones that exhibited at least 10-fold higher ELISA signal in target protein coated plates compared to BSA coated plates. Individual clones were selected after 4 and 5 steps of selection to bind to human DR5 or human HER-2. Specific ligands were subjected to sequence analysis. Specific ligands were also analyzed using a spot ELISA, ELISA specificity, and HER-2 affinity assay using methods such as those described herein. (See, Figure 11.) As indicated in Figure 10, YSGR-AD libraries produce specific ligands against both target proteins.

Of the 240 clones identified that specifically bind to human HER-2, 106 of them had unique CDR sequences (See Figure 11). Unique sequences fell into 3 categories: 1) 6-7 residue after CDRH3; 2) eight residue in the CDRH3 sequences; and 3) CDR average length in the tyrosine, serine and glycine sequences in the sequence.

The anti-HER-2 heavy chain variable domain demonstrated a preference for short CDRH3 sequences (e.g., 6-7 amino acids at positions corresponding to 95 to 100a) not included in the oligo library (See Figure 12). CDRH3 exhibits a conserved N-terminal tyrosine residue at position 95. The other conserved position is found at position 99, which is predominantly glycine. Consensus sequences are displayed for CDRL3: QQSYYX4PST (SEQ ID NO: 587); CDRH1-.GFSIX2X3SYIH (SEQ ID No: 588); and CDRH2:

SIYPX3SGYTSYADSKVG (SEQ ID No: 589), wherein X represents an amino acid deposition for which a consensus residue has not been identified and where the X position in each CDR is Y or S.

CDRH3-containing eight-amino acid heavy chain variable domains have conserved glycines at the end of the CDRH3 N and C-termini (at positions 95 and 100a). Position 98 is also preserved as a tyrosine. Position 99 with an 8 amino acid CDHR3 demonstrates a preference for a small amino acid such as G, S, A, or T. This position is followed by a large amino acid at position 100, such as R, H, Y, and W. Consensus sequences are shown by CDRH1: GFX1ISYSSIH (SEQ ID No590); and CDRH2.SIYPX3YGX5TX6YADSKVG (SEQ ID NO: 591), where X represents an amino acid position for which a consensus residue has not been identified and is Y or S.

Analysis of the medium length heavy chain variable domain (e.g., about 12 to 14 amino acids) of the CDRH3 region provides a consensus CDRH3 sequence; XEX2X3X4YYSYYX10GX12X13X14DY (SEQ ID NO: 592), where X represents an amino acid position for which a consensus residue has not been identified and where position X1 is selected from Y, S and R, X2 is selected from Y and S; X3 is selected from G, Y and S; X4 is selected from G, Y and S; X4 is selected from Y, S, R and G; X10 is selected from Y, S and G, X12 is selected from Y, S and G; X13 is selected from G and A and X14 is selected from I, F, M and L (see, Figure 14). As CDRH3 forms a loop, the consensus for CDRH3 was developed by transferring the sequence to some clones of more than two amino acids, so that position 95 of the sequence could align with position 97 of the reference sequence, which in this case was clone sequence 52. The consensus sequence is also demonstrated for CDRH1: GFX1ISSSS1H (SEQ ID No: 593); eCDRH2: X1IX2PSSGYTX6YADSKVG (SEQ ID NO: 594), wherein X represents an amino acid position for which a consensus residue has not been identified and is Y or S.

As described in Figure 11B, several of the HER-2-linked clones had IC50 between 0.1 to 10 nM. For the most part, these high affinity ligands had little or no cross-reaction with other antigens such as VEGF, DR5, insulin, neutravidine, human growth hormone or IGF-1.

As indicated in Figure 10, 144 Fabs-expressing clones were identified as being specific binders for human DR5. Following analysis identified 18 unique amino acid sequences of these144 clones, shown in Figure 15.

The IC50 of each of these ligands was determined by ELISA phage competition, followed by incubation in 96-NUNCMaxisorp Immunoplates coated overnight wells at 4 ° C with hDR5-ECD (5 µg / ml) and BSA-blocked. Fabs exhibited by Phages were multiplied into E. coliXU-blue with the addition of M13-K07 phage helper. After governight growth at 37 ° C in 2YT medium, the phages were concentrated by precipitation in PEG / NaCl and resuspended in phosphate buffered saline buffer (PBS), 0.5% (w / v) BSA, 0.1% (v / v) Tween 20 (PBT buffer). Phages were serially diluted in PBT buffer and binding was measured to determine phage concentration giving a saturation signal of - 50%. A fixed unsaturated phage concentration was preincubated for 2h in hDR5-ECD serial dilutions and then transferred. for analysis of hDR5-ECD coated plates. After 15 min incubation, the plates were washed with PBS, 0.05% Tween 20 and incubated for 30 min with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer). The plates were washed, developed with TMB substrate, and blotted with 1.0 M H3PO4, and read spectrophotometrically at 450 nm. The affinity deletion of anti-hDR5 ligands

The binding affinity of anti-hDR5 ligands was determined as IC50 values defined as the concentration of hDR5-ECD that blocked 50% of phage binding to immobilized hDR5-ECD.

Clones were analyzed by binding to the human DR5 extracellular domain (SEQ ID No: 595). Ligands with the smallest IC50 have predominantly syrine on CDRH1 and arginine on CDRH3 at positions 96 and 99. CDRH3 region heavy chain variable domain analysis provides a consensus sequence of CDRH3: YRX3YRYGX8X9X10GSYX14X15DY (SEQ ID NO: 596), where X3 is selected from Y, S, R, P and G; X8 is selected from R, Y and S; X9 is selected from G, Y and S; X10 is selected from S, Y and R; X14 is selected from G and A and X15 is selected from L and F, where X represents an amino acid position for which a consensus residue has not been identified, (see, Figure 16). Consensus consequence is also demonstrated for CDRL3: QQXIX2X3SPST (SEQ ID No: 597), where X1, X2, and X3 are Y or S; CDRH2: X1ISPX3X4GYTX6YADSKVG (SEQ ID NO: 599), where X represents an amino acid deposition for which a consensus residue has not been identified and is X or Y. Consensus sequences can be used, inter alia, to construct new variable domain libraries of antibodies. In CDRH3, amino acids at positions 97, 100b, 100c, 100h and 100i may contribute to the highest affinity.

Clones were also analyzed by binding to DR5 by using the competition phage ELISA described above. Several clones were isolated from the YSGR-A-D library that bound to both human DR5 and murine DR5, although binding to murine DR5 was much less affinity (see Figure 17). This library is provided for the isolation of antibodies that can bind to both human DR5 and murine DR5 indicating that the ligands were identified as unique compared to a total of randomized CDRH3 (all twenty amino acids) and a YS CDRH3 library. By modifying the diversity of amino acids allowed at each position, it can provide antibodies that bind to different epitopes and have unique biological functions. Anti-DR5 antibodies that bind to both human CDRs and murine CDRs may seal different epitopes than those anti-DR5 antibodies from previously developed libraries.

exemplq3

DR5 Ligand Analysis

The Apo 2L ligand binding site for human DR5 has been previously mapped and the crystal structure for the binding site has been determined (see, Hymowitz et al, Molecular Cell 4: 564 (1999); and WO01 / 19861). The crystal structure and models can be used to map binding of anti-DR5 antibodies.

Previous studies have identified antibodies that bind to human DR5. These antibodies are called BDF1 and YSD1. The BDF1 antibody was isolated from a library in which CDRs were randomized to all 20 amino acids and having the CDR sequences: 1) deIGKSGIH CDRH1 sequence (SEQ ID No: 600); 2); VAVIYPHDGNTAYA CDR2 Sequence (SEQID No: 601); and 3) CDRH3 sequence of RLALVRMWMD (SEQ ID No: 602). Antibody YSD1 has been isolated from a library in which CDR positions have been tyrosine- and serine-variant and has a CDRH3 sequence of YSSYYSYYYSSSSYSY (SEQ ID NO: 603). with the Apo 2L ligand, which is predominantly at the C-terminus (50s loop amino acids, e.g. amino acids, 50-65 loop 90 amino acids, e.g. DR5 amino acids 91 to 104). A model demonstrating the binding of the CDRH3 regions of each of these antibodies is shown in Figure 18. The BDF1 and YSD1 CDRH3 overlap and form a preferred DR5 hot spot. YSD1 CDRH3 binding is tyrosine mediated and BFD1 binding is mediated by LAL sequence YSD 1 binding in DR5 involves leucine, glutamine, alanine, phenylalanine, arginine and DR5 residues.

Example 4

Construction Of Fab Libraries Displayed By Phage With Debris From CDRH1, H2 And L3 Enriched In Ser And Wing. Cys. ESP, Gly, Ile, Leu. Asn,

Pro. Arg. Thr. Trp or tyr

Phage-displayed Fab libraries were constructed using a phagemid vector, Fab-C, which resulted in the display of Fabbivalent molecules dimerized by a free cysteine inserted between the heavy-chain Fab and the C-terminal domain of the capsid secondary protein gene-3 ( P3C) as previously described in Example 1.

Twelve libraries were built: SAH3, SCH3, SFH3, SGH3, SLH3, SNH3, SPH3, SRH3, STH3, SWH3, and SYH3. Libraries were constructed with randomized residues on all three CDR3 light and heavy chain CDRs. Each library was randomized at CDRL3 positions 91-94 and 96, CDRH1 positions 28 and 30-33, CDRH2 positions O, 52-54, 56 and 58 and CDRH3 positions 95-100, 100a to 100m. The type and ratio of amino acids allowed for each of the randomized positions are illustrated in Figures 19A-19B. In addition, the length of CDRH3 was varied using oligonucleotides that replaced the six wild type codons at positions 95 and 100 with 4 to 10 codons. The type and ratio of amino acids allowed at these positions were the same as those described in Figures 19A-19B for positions 95-100 of CDRH3. Libraries were constructed using the Kunkel method (Kunkel, et al., Methods Enzymol. (1987), 154, 367-382), with previously described methods (Sidhu, SS, et al., Methods Enzymol. (2000), 328, 333-363). A single "stop template" version of the Fab display vector Fab-C was used to generate all four libraries as described in Example 1.

Mutagenic oligonucleotides with degenerating codons in the positions to be diversified were used to simultaneously (a) introduce diversity into CDR and (b) repair stop codons. The sequences of these mutagenic oligonucleotides are shown in Figures 20A-20L. For all libraries, diversity was introduced into CDR-H1, CDR-H2 and CDR-H3 with oligonucleotides H1, H2 and L3, respectively (SEQ ID Nos. :).

For the SAH3 library, a CDR-H3 diversity was introduced with an equimolar mixture of H3-SA4, H3-SA5, H3-SA6, H3-SA7, H3-SA8, H3-SA10, H3-SA11 oligonucleotides. , H3-SA12, H3-SA13, H3-SA14, H3-SA15, H3-SA16, and H3-SA17 (SEQ ID Nos: 621-634).

For the SCH3 library, a diversity of CDRH3 with an equimolar mixture of oligonucleotides H3-SC4, H3-SC5, H3-SC6, H3-SC8, H3-SC9, H3-SC10, H3-SC11, H3- SC12, H3-SC13, H3-SC14, H3-SC15, H3-SC16, and H3-SC17 (SEQ ID NOS: 635-648).

For the SFH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of oligonucleotides H3-SF4, H3-SF5, H3-SF6, H3-SF7, H3-SF8, H3-SF10, H3-SF11 , H3-SF12, H3-SF13, H3-SF14, H3-SF15, H3-SF16, and H3-SF17 (SEQ ID Nos: 649-662).

For the SGH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-SG4, H3-SG5, H3-SG6, H3-SG7, H3-SG8, H3-SG10, H3-SG11 oligonucleotides. , H3-SG12, H3-SG13, H3-SG14, H3-SG15, H3-SG16, and H3-SG17 (SEQ ID Nos: 663-676). For the SIH3 library, a diversity of CDR-H3 with a equimolar mixture of oligonucleotides H3-SI4, H3-SI5, H3-SI6, H3-SI7, H3-SI8, H3-SI10, H3-SI11, H3-SI12, H3-SI13, H3-SI14, H3- SI15, H3-SI16, and H3-SI17 (SEQ ID NOS: 677-690).

For the SLH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-SL4, H3-SL5, H3-SL6, H3-SL7, H3-SL8, H3-SL10, H3-SL11 oligonucleotides. , H3-SL12, H3-SL13, H3-SL14, H3-SL15, H3-SL16, and H3-SL17 (SEQ ID Nos: 691-704).

For the SNH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of oligonucleotides H3-SN4, H3-SN5, H3-SN6, H3-SN7, H3-SN8, H3-SN10, H3-SN11 , H3-SN12, H3-SN13, H3-SN14, H3-SN15, H3-SN16, and H3-SN17 (SEQ ID NOS: 705-718).

For the SPH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-SP4, H3-SP5, H3-SP6, H3-SP7, H3-SP8, H3-SP10, H3-SP11 oligonucleotides. , H3-SP12, H3-SP13, H3-SP14, H3-SP15, H3-SP16, and H3-SP17 (SEQ ID NOS: 719-732).

For the SRH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-SR4, H3-SR5, H3-SR6, H3-SR7, H3-SR8, H3-SR10, H3-SR11 oligonucleotides. , H3-SR12, H3-SR13, H3-SR14, H3-SR15, H3-SR16, and H3-SR17 (SEQ ID NOS: 733-746).

For the STH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of H3-ST4, H3-ST5, H3-ST6, H3-ST7, H3-ST8, H3-ST10, H3-ST11 oligonucleotides. , H3-ST12, H3-ST13, H3-ST14, H3-ST15, H3-ST16, and H3-ST17 (SEQ ID Nos: 747-760).

For the SWH3 library, a diversity of CDR-H3 was introduced with an equimolar mixture of oligonucleotides H3-SW4, H3-SW5, H3-SW6, H3-SW7, H3-SW8, H3-SW10, H3-SW11 , H3-SW12, H3-SW13, H3-SW14, H3-SW15, H3-SW16, and H3-SW17 (SEQ ID Nos: 761-774). For the SYH3 library, a diversity of CDR-H3 with a equimolar mixture of oligonucleotides H3-SY4, H3-SY5, H3-SY6, H3-SY7, H3-SY8, H3-SY9, H3-SY11, H3-SY12, H3-SY13, H3-SY14, H3- SY15, H3-SY16, and H3-SY17 (SEQ ID Nos: 775-788).

Mutagenic oligonucleotides for all formatted CDRs to be incorporated simultaneously into a single demutagenesis reaction, thus the simultaneous incorporation of all mutagenic oligonucleotides resulted in the introduction of the projected diversity at each position, while simultaneously repairing all TAA stop codons. Thus, an open reading frame was generated to encode a Fab library fused to a homodimerizing cysteine bridge and P3C. Following mutagenesis, the twenty libraries were combined to create a single library, called the SXH3 library.

Mutagenesis reactions were electroporated into E. coli SS320 (Sidhu et al., Supra). Transformed cells were grown overnight in the presence of M13-K07 phage helper (New England Biolabs, Beverly, MA) to produce phage particles that encapsulate phagemid DNA and display Fab fragments on their surfaces. The combined library contains more than 3 x 1010 unique members.

Example 5

Selection of Specific Antibodies from SXH3 Libraries NaivesPhage from the SXH3 library (described in Example 4, above) were cycled by ligation selection steps to enrich human HER-2 clones. Selections were made using binding methods described above (Sidhu et al, Supra).

Maxisorp Immunoplates 96-well NUNC plates were coated and incubated overnight at 4 ° C with 5 µg / ml target protein (human HER-2) and blocked for 2 hours with a PBT solution (additionally containing 0.2% BSA and 0% phosphate buffer). 0.05% Tween 20 (Sigma)). After overnight growth at 37 ° C, the phages were concentrated by PEG / NaCl precipitation and resuspended in PBT as previously described (Sidhu et al., Supra). Phage solution (about 1012 phage / mL) was added to the coated Immunoplate plate. After two hours of incubation to allow phage to bind, the plates were washed ten times with PBT. The bound phage were eluted with 0.1 M HCl for ten minutes and the eluents were neutralized with 1.0 M Tris base. The eluted phage were amplified in E. coli XL-1-blue and used for further selection steps.

The libraries were subjected to six selection steps against the target protein. Individual clones from each selection step were grown in 96-well wells in 500 µl of 2YT broth supplemented with carbenicillin and M13-K07. The culture supernatant was used directly on ELISA phage (Sidhu et al, Supra) to detect phage-displayed Fabs that bound on target protein coated plates, but did not bind to BSA coated plates. Specific ligands were defined as those phage clones that exhibited an ELISA signal of at least 10-fold greater on target protein coated plates compared to BSA coated plates. Individual clones were selected after 4, 5 and 6 selection steps to bind to human HER-2. Specific ligands were subjected to sequence analysis. As shown in Figure 21, SXH3 libraries produce specific protein binding ligands.

Of the 72 clones identified that specifically bind HERHER-2, 27 of them had unique CDR sequences (See Figure 21A). Unique consequences fell into 3 categories: a) CDR sequences with randomized positions limited to two Tyr / Ser parts (clones Nos: B1-5 and B28); b) CDR sequences with randomized positions limited to Trp / Ser (clones Nos: B6-24); and c) CDR sequences with randomized positions limited to Phe / Ser (clones Nos: B25-27). These clones were also highly specific for HER2 and did not cross-react with the other five control proteins, human VEGF, human DR5, human insulin, neutravidine, human IGF-1 or HGH (see Figure 21B). The inhibitory concentration for each clone is shown in Figure 21B.

A phage ELISA was used to test the ability of all clones to cross-react with a panel of six different antigens from the target antigen. Phages were produced in 96-well plates as described and the supernatant was diluted 3-fold in PBT buffer. The diluted phage supernatant was transferred to plates coated with human VEGF, human HER2, human DR5, human insulin, neutravidine, human IGF-1, HGH or BSA and incubated for one hour under gentle shaking at room temperature. The plates were washed in PBS including 0.05% Tween 20 and incubated for 30 min with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (phamacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories), and blotted out with 1.0M H3PO4. Absorbance was determined spectrophotometrically at 450 nm. Weak binding affinity was defined as a signal from about 0.2 to 2.0, and weak binding affinity was defined as a signal of about 2.0. Results for HER2 binding clones are shown in Figure 21B. As shown in Figure 25, of the isolated SXH3 clones, the S: R clones exhibited a higher mean nonspecific deletion (OD 0.5-0.6 at 450 nm by the ELISA test), while the S: W, S clones : Y, and S: F exhibited low or medium levels of non-specific binding (OD 0-0.1 at 450 nm by ELISA).

A phage ELISA competition test was used to estimate HER-2 binding affinity for phage displayed Fabs. Phages were produced in 96-well plates as described above, the supernatant was serially diluted in PBT buffer and then incubated in HER2 coated plates for 15 minutes. The plates were washed in PBS including 0.05% Tween 20 and incubated for 30 minutes with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (phamacia). The plates were washed, grown with tetramethylbenzidine (TMB) substrate (Kirkegaard and PerryLaboratories), and blotted out with 1.0 M H3PO4. Absorbance was determined spectrophotometrically at 450 nm to determine the concentration of phage by a ~ 50% saturation signal. A phage-fixed unsaturated concentration was diluted twice in PBT buffer containing serial dilutions in duplicates of HER-2 protein from 250 nM HER-2 to 0.12 nM HER-2. The mixtures were incubated for one hour under gentle shaking at room temperature and transferred to HER2 coated plates and then incubated for 15 minutes. The plates were washed and treated exactly as described above. Binding affinity was estimated as IC50 (defined as antigen concentration that blocked 50% of phage binding in immobilized antigens). Results are shown in Figure 21B.

Example 6

Construction Of Fab Libraries Displayed By Phage With DeCDR Waste Enriched In Ser And Phe. Arg. Trp or tyr

Phage-displayed Fab libraries were constructed using a phagemid vector, Fab-C, which resulted in the display of Fabbivalent molecules dimerized by a free cysteine inserted between the heavy-chain Fab and the C-terminal domain of the capsid secondary protein gene-3 ( P3C) as previously described in Example 1.

Four libraries were built: SFH3, SRH3, SWH3 andSYH3. Libraries were constructed with randomized residues on all three CDRs and the light chain CDR3. Each library was randomized to CDRL3 positions 91-94 and 96, CDRH1 positions 28 and 30-33, CDRH2 positions O, 52-54, 56 and 58 and CDRH3 positions 95-100, 100a to 100m. The amino acid type and ratio allowed for each of the randomized positions are illustrated in Figure 22. In addition, the length of the CDRH3 was varied using oligonucleotides that replaced the six wild type codons at positions 95 and 100 with 4 to 17 codons. The type and ratio of amino acids allowed at these positions were the same as those described in Figure 22 for positions 95-100 of CDRH3.

Libraries were constructed using the Kunkel method (Kunkel, TA, Roberts, JD & Zakour, RA, Methods Enzymol. (1987), 154, 367-382), with previously described methods (Sidhu, SS, Lowman, HB, Cunningham, BC & Wells, JA, Methods Enzymol (2000), 328, 333-363). A single "stop template" version of the Fab display vector, Fab-C was used to generate all four libraries as described in Example 1.

Mutagenic oligonucleotides with degeneracy codons in the positions to be diversified were used to simultaneously (a) introduce diversity into CDR and (b) repair stop codons. The sequences of these mutagenic oligonucleotides are shown in Figures 20 and 23. For all SF-surface libraries, diversity was introduced in CDR-L3, CDR-H1 and CDR-H2 with L3-SF, H1-SF and H2-SF oligonucleotides, respectively (SEQ IDNos :) (Figure 23) and diversity was introduced into CDR-H3 with an equimolar mixture of oligonucleotides H3-SF4, H3-SF5, H3-SF6, H3-SF7, H3-SF9, H3-SF10, H3- SF11, H3-SF12, H3-SF13, H3-SF14, H3-SF15, H3-SF16, and H3-SF17 (SEQ ID Nos: 649-662) (Figure 20 C).

For the SR-surface library, diversity was introduced into CDR-L3, CDR-H1 and CDR-H2 with L3-SR, H1-SR and H2-SR oligonucleotides, respectively (SEQ ID Nos.) (Figure 23) and diversity. was introduced into CDR-H3 with an equimolar mixture of oligonucleotides H3-SR4, H3-SR5, H3-SR6, H3-SR7, H3-SR8, H3-SR9, H3-SR10, H3-SR11, H3-SR13 , H3-SR14, H3-SR15, H3-SR16, and H3-SR17 (SEQ ID Nos: 733-746) (Figure 201).

For the SW-surface library, diversity was introduced into CDR-L3, CDR-H1 and CDR-H2 with L3-SW, H1-SW and H2-SW oligonucleotides, respectively (SEQ ID Nos: 747-760) (Figure 23) and diversity was introduced into CDR-H3 with an equimolar mixture of oligonucleotides H3-SW4, H3-SW5, H3-SW6, H3-SW7, H3-SW8, H3-SW9, H3-SW11, H3-SW12, H3 -SW13, H3-SW14, H3-SW15, H3-SW16, and H3-SW17 (SEQ IDNs: 761-774) (Figure 20K).

For the SY-surface library, diversity was introduced into CDR-L3, CDR-H1 and CDR-H2 with oligonucleotides L3, H1 and H2, respectively (SEQ ID Nos. :) (Figure 20A) and diversity was introduced into CDR-H3 with an equimolar mixture of oligonucleotides H3-SY4, H3-SY5, H3-SY6, H3-SY7, H3-SY8, H3-SY10, H3-SY11, H3-SY12, H3-SY13, H3-SY14, H3 -SY15, H3-SY16, and H3-SY17 (SEQ ID Nos: 775-788) (Figure 20L).

Mutagenic oligonucleotides for all formatted CDRs to be incorporated simultaneously into a single demutagenesis reaction, thus the simultaneous incorporation of all mutagenic oligonucleotides resulted in the introduction of the projected diversity at each position, while simultaneously repairing all TAA stop codons. Thus, an open reading frame was generated to encode a Fab library fused to a homodimerizing cysteine bridge and P3C. Following mutagenesis, the twenty libraries were combined to create a single library, called the SX-surface library.

Mutagenesis reactions were electroporated into E. coli SS320 (Sidhu et al, Supra). Transformed cells were grown overnight in the presence of M13-K07 phage helper (New England Biolabs, Beverly, MA) to produce phage particles that encapsulate phagemid DNA and display Fab fragments on their surfaces. The combined library contains more than 3 x 1010 unique members.

Example 7

Selecting Specific Antibodies from SX-SurfaceNaives Libraries

SX-Surface library phage (described in Example 6, above) were cyclized by ligation selection steps to enrich human HER-2 ligand clones. Selections were performed using the binding methods described above (Sidhu et al, Supra).

Maxisorp Immunoplates 96-well NUNC plates were coated and incubated overnight at 4 ° C with 5 µg / ml target protein (human HER-2) and blocked for 2 hours with a PBT solution (Sigma). After overnight growth at 37 ° C, the phages were concentrated by PEG / NaCl precipitation and resuspended in PBT as described above (Sidhu et al, Supra). Phage solution (about 1012 phage / mL) was added to the coated Immunoplate plate. After two hours of incubation to allow phage to bind, the plates were washed ten times with PBT. The bound phage were eluted with 0.1 M HCl for ten minutes and the eluents were neutralized with 1.0 M Tris base. The eluted phage were amplified in E. coli XL-1-blue and used for further selection steps.

The libraries were subjected to six selection steps against the target protein. Individual clones from each selection step were grown in 96-well wells in 500 µl of 2YT broth supplemented with carbenicillin and M13-K07. The culture supernatant was used directly on ELISA phage (Sidhu et al, Supra) to detect phage-displayed Fabs that bound on target protein coated plates, but did not bind to BSA coated plates. Specific ligands were defined as those phage clones that exhibited at least 10-fold greater ELISA signal in target protein coated plates compared to BSA coated plates. Individual clones were selected after 4, 5 and 6 selection steps to bind to human HER-2. Specific ligands were subjected to sequence analysis. As indicated in Figure 21, SX-Surface libraries produce specific protein binding ligands.

Of the 81 clones identified that specifically bind HERHER-2, 27 of them had unique CDR sequences (See Figure 24A). Unique consequences fell into two categories: (a) CDR sequences with randomized positions limited to the Tyr / Ser binary (clones Nos: G49-61); (b) CDR sequences with random positions limited to binary Trp / Ser (clones Nos: G29-48). Tyr / Ser clones were highly HER2 specific and did not cross-react with the other five control proteins, human VEGF, human DR5, human insulin. , neutravidine, human IGF-1 or HGH (see, Figure 24B). The inhibitory concentration for each clone is shown in Figure 24B.

A phage ELISA was used to test the ability of all clones to cross-react with a panel of six different antigens from the target antigen. Phages were produced in 96-well plates as described and the supernatant was diluted 3-fold in PBT buffer. The diluted phage supernatant was transferred to plates coated with human VEGF, human HER2, human DR5, human insulin, neutravidine, human IGF-1, HGH or BSA and incubated for one hour under gentle shaking at room temperature. The plates were washed in PBS including 0.05% Tween 20 and incubated for 30 min with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (phamacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories), and blotted with 1.0 M H3PO4. Absorbance was determined spectrophotometrically at 450 nm. Weak binding affinity was defined as a signal from about 0.2 to 2.0 and strong binding affinity was defined as a signal of about 2.0. Results for the SX-surface clones are shown in Figure 24B. As shown in Figure 27, of the isolated clones of the SX-surface library, S.Re S: W clones exhibited a higher nonspecific binding mean (OD 0.50- 0.6 and approximately OD 4.0 respectively at 450 nm by the ELISA test), while clones S: Y and S: F exhibited low or medium non-specific deletion (OD 0-0.1 at 450 nm by the ELISA test). ELISA).

A phage ELISA competition test was used to estimate HER-2 binding affinity for phage displayed Fabs. Phages were produced in 96-well plates as described above, the supernatant was serially diluted in PBT buffer and then incubated in HER2 coated plates for 15 minutes. The plates were washed in PBS including 0.05% Tween 20 and incubated for 30 minutes with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (phamacia). The plates were washed, grown with tetramethylbenzidine (TMB) substrate (Kirkegaard and PerryLaboratories), and blotted with 1.0 M H 3 PO 4. Absorbance was determined spectrophotometrically at 450 nm to determine the concentration of phage by a ~ 50% saturation signal. A phage-fixed unsaturated concentration was diluted twice in PBT buffer containing serial dilutions in duplicates of HER-2 protein from 250 nM HER-2 to 0.12 nM HER-2. The mixtures were incubated for one hour under gentle shaking at room temperature and transferred to HER2 coated plates and then incubated for 15 minutes. The plates were washed and treated exactly as described above. Binding affinity was estimated as IC50 value (defined as the antigen concentration that blocked 50% of phage binding in immobilized antigens). Results are shown in Figure 24B.

Based on this analysis, analysis of HER2 binding clones from the SXH3 library (Example 6), and from the YSGR-AD library (Example 1), soluble Fab proteins from three clones (clone Nos .: 42 (YSGR-A) and B11 (SXH3) and G54 (SX-surface)), were purified and subjected to surface plasmon resonance assay using for human HER2 binding. BlAcore® data were obtained according to Chen et al. J.Mol. Biol. (1999), 293 (4): 865-81. Briefly, the binding affinity of human HER2 purified Fabs were calculated from the association and dissociation constants measured using the BlAcore ® -A100 surface plasmon resonance system (BIACORE, Inc., Piscataway, NJ). OHER-2 was covalently coupled to a biosensor chip at two different concentrations using N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysucinimide (SNS) according to the instructions of the supplier (BIACORE, Inc. , Piscataway, NJ). HER-2 was diluted with 10mM sodium acetate, pH 5.0, to approximately 2.5 or 5 µg / ml. HER2 aliquots were injected at a flow rate of 5 µl / minute to reach approximately 50 to 170 units. coupled protein response time (UK). A 1M ethanolamine solution is added as a blocking agent. For kinetic mediation, serial dilutions in duplicates of Fab were injected into HBT at 25 ° C at a flow rate of about 10 µl / min over each cell flow. The association velocity (kon) were calculated using a binding curve using the BlAcore EvaluationSoftware package (BIACORE, Inc., Piscataway, NJ) using the two-point local and global assembly and combining data from both cell streams A dissociation equilibrium constant (Kp) is calculated as k0ff / kon correlation. The BlAcore ® data are summarized in Figures 26A and B. Occlone B1L had a 1.9 x 10 6 M "1 S" 1 ka, a 1.7 x 10 -3 3 k "1 kd and a 890 nM KD. for the clone B1I experiment was 19 RU, and Rmax2 for the clone B1I experiment was 29 RU. (Figure 26A) Clone G54 had a 2.0 x 105 M "1 S" 1 umka, one kdde 2, 2 x 10'3 S'1 and an 11 nM KD. The Rmaxi for the G54 clone experiment was 21 RU and Rmax2 for the G54 clone experiment was 34 RU. (Figure 26A) Clone YSGR-A-42 had a 2.7 x 10 6 M "1S" 1 ka, a 1.5 x 10 "3 s" 1 Kd, and a 570 pM KD. The Rmaxi for the clone 42 experiment was 25 RU, and Rmax2 for the clone42 experiment was 38 RU. (Figure 26b) The tryptophan-containing clone (B1I) had a correspondingly lower kon and KD velocity than the containing-tyrosine clone (G54).

To study binding of anti-HER2 antibodies to HER2 expressed in mammalian cells, binding of purified Fab protein from clones 42 (YSGR-A), B1I (SXH3), G54 (SX surface), and G37 (SX surface) cells NR6 fibroblasts that overexpress HER2 (NR6-HER2) were studied by flow cytometry. One million NR6-HER2 cells were incubated with 10 µg / ml Fab for 1 hour, followed by incubation with aAlexa488-murine anti-human IgG Antibody conjugate for 1 hour. As a negative control, Fab bound to non-expressing NR6 cells were studied. As a positive control, Fab 4D5 was used. As shown in Figure 27, clones 42, B1I, G54, G37 specifically bind to HER2 on NR6 cells.

An ELISA competition test was used to assess binding binding with Omnitarg, Herceptin and among some types of IgG clonesHER2 (see, Figure 28 for CDR sequences of relevant clones). Biotinylated HER2 protein was serially diluted from 200 nM to 0.39 mM in PBT buffer and then incubated in purified IgG comprotein coated plates for 15 minutes. The plates were washed in PBS including 0.05% Tween 20 and incubated for 30 minutes with horseradish peroxidase / anti-M13 antibody conjugate (1: 5000 dilution in PBT buffer) (phamacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories), and washed with 1.0 M H3PO4. Absorbance was determined spectrophotometrically at 450 nm to determine the concentration of biotinylated HER2 giving a saturation signal of ~ 50%. A fixed subsaturated concentration of biotinylated HER2 protein was diluted twice in PBP buffer containing 100 mM of purified IgG proteins. The mixtures were incubated for one hour under gentle shaking at room temperature and transferred to plates coated with purified IgG proteins and then incubated for 15 minutes. The plates were washed and treated exactly as described above. As shown in Figure 29, none of the HER2 IgG ligands blocked binding of biotinylated HER2 with either Omnitarg or Herceptin. IgGs blocked the bond between them in two groups. A group consisting of clones B1I, G37, G54, and YSGR-A-42 competed for the same epitope and blocked binding to the biotinylated HER2, which had previously been incubated with any of these clones. A second group consisting of clones YSGR-A-27, B27, G43 andYSGR-D-104 competed for the same epitope to HER2 and blocked binding to biotinylated HER2. All group one clones have higher binding affinity than group two clones.

All publications (including patents and patent applications) cited herein are incorporated in their entirety by reference.

Table 2

<table> table see original document page 263 </column> </row> <table> EDGRDCISCKYGQDYSTHWKDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREED

SPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPASP

CSLS (SEQ ID No: 595)

DR5 polypeptide humanomeqrgqnapa asgarkrhgp gpreargarp glrvpktlvl vvaavlllvs aesalitqqd lapqqraapqqkrsspsegl cppghhised grdcisckyg qdysthwndl Ifclrctrcd sgevelspct ttrntvcqceegtfreedsp emcrkcrtgc prgmvkvgdc tpwsdiecvh kesgiiigvt vaavvlivav fvcksllwkkvlpylkgics ggggdpervd rssqrpgaed nvlneivsil qptqvpeqem evqepaeptgvnmlspgese hllepaeaer sqrrrllvpa negdptetlr qcfddfadlv pfdsweplmr klglmdneikvakaeaaghr dtlytmlikw vnktgrdasv htlldaletl gerlakqkie dhllssgkfm ylegnadsal s (SEQ ID NO: 604)

Murine DR5-ECD

GLQRPEESPSRGPCLAGQYLSEGNCKPCREGIDYTSHSNHSLDSCILCTVCKEDKWETR

CNTrTNTVCRCKPGTFEDKDSPEICQSCSNCTDGEEELTSCTPRENRKCVSKTAWASWHK

SEQ ID No: 605)

Apo-2L Polypeptide Sequence

1

MetAlaMetMetGluValGInGlyGlyProSerLeuGlyGInThrCysValLeulIeValIlePheThr

ValLeuLeuGInSerLeuCys

31

ValAlaValThrTyrValTyrPheThrAsnGluLeuLysGInMetGInAspLysTyrSerLysSerGI

ylleAlaCysPheLeuLysGIu

61

AspAspSerTyrTrpAspProAsnAspGluGluSerMetAsnSerProCysTrpGInValLysTrpGlnLeuArgGInLeuVaLArgLys91

MetlleLeuArgThrSerGIuGluThrlIeSerThrValGInGluLysGInGInAsnlIeSerProLeuValArgGluArgGlyProGln

121

ArgValAlaAIaHislleThrGIyThrArgGlyArgSerAsnThrLeuSerProAsnSerLysAsnGluLysAlaLeuGlyArgLys

151

IleAsnSerTrpGluSerSerArgSerGIyHisSerPheLeuSerAsnLeuHisLeuArgAsnGlyGIuLeuVallleHisGluLysGly

181

PheTyrTyrlIeTyrSerGInThrTyrPheArgPheGInGluGlulleLysGluAsiiThrLysAsnAspLysGInMetValGInTyrlIe

211

TyrLysTyrThrSerTyrProAspProlleLeuLeuMetLysSerAlaArgAsnSerCysTrpSerLysAspAlaGluTyrGIyLeuTyr

241

SerlIeTyrGInGlyGlyllePheGluLeuLysGluAsnAspArgIlePheValSerValThrAsnGIuHisLeulIeAspMetAspHis

271

GluAlaSerPhePheGlyAlaPheLeuValGIy (SEQ ID No: 606)

amino acid sequence 114-281 of Apo-2LVRERGPQRVA AHITGTRGRS NTLSSPNSKN EKALGRKINS WESSRSGHSFLSNLHLRNGE LVEHEKGFYY IYSQTYFRFQ EEIKENTKND KQMVQYIYKYTSYPDPELLM KSARNSCWSK DAEYGLYSIY QGGEFELKEN DRIFVSVTNEHLIDMDHEAS FFGAFLVG (SEQ ID NO: 607) sequence .Listagem

<110> Sidhu, Sachdev S.Birtalan, SaraFellouse, Frederic

<120> LINKING POLYPEPTIDES AND THEIR USES

<130> 11669.291USU1

<140> NEW DEPOSIT <141> 12-01-2006

<150> 60 / 742,185 <151> 12-02-2005

<150> 60 / 805,553 <151> 22-06-2006

<160> 1013

<170> Patentln version 3.3

<210> 1

<211> 109

<212> PRT

<213> Artificial

<220>

<223> Synthetic 4D5 light chain variable domain <400> 1

Asp lie Gln Met Thr Gln Ser Pro Ser Ser Ser Ala Ser Ser Gly1 5 10 15

Asp Arg Going Thr Lie Thr Cys Arg Wing Be Gln Asp Going Asn Thr Ala20 25 30

Go Trp Wing Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu lie35 40 45

Tyr Ser Ala Ser Ser Ser Tyr Ser Gly Go Pro Ser Arg Phe Ser Gly50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gln Pro65 70 75 80

Glu Asp Phe Ala Thr Thr Tyr Cys Gln His Tyr Thr Thr Pro Pro 90 90 95

Thr Phe Gly Gln Gly Thr Lys Goes Glu lie Lys Arg Thr100 105

<210> 2 <211> 120 <212> PRT

<213> Artificial

<220>

<223> Synthetic 4D5 heavy chain variable domain <400> 2

Glu Goes Gln Leu Goes Glu Be Gly Gly Gly Leu Goes Gln Pro Gly Gly1 5 10 15

Ser Leu Arg Leu Ser Cys Wing Ala Ser Gly Phe Asn lie Lys Asp Thr20 25 30

Tyr lie His Trp Goes Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Vai35 40 45

Wing Arg lie Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Wing Asp Ser Vai50 55 60

Lys Gly Arg Phe Thr Lie Be Wing Asp Thr Be Lys Asn Wing Wing Tyr65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Go Tyr Tyr Cys85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Met Asp Tyr Trp Gly Gln100 105 110

Gly Thr Leu Will Thr Will Be Ser115 120

<210> 3

<211> 35

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 3

Gly Arg Met Lys Gln Leu Glu Asp Lys Goes Glu Glu Leu Leu Ser Lys15 10 15

Asn Tyr His Leu Glu Asn Glu Go Ala Arg Leu Lys Lys Leu Go Gly20 25 30

Glu Arg Gly35

<210> 4

<211> 11

<212> PRT

<213> Artificial <220>

<223> Synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is independently from Y, G, S, R, A, D, E, F, H, I, K, L,

M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE

<222> (2). . (2)

<223> X is independently Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (3). . (3)

<223> X is independently Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (4). . (4)

<223> X is independently Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (5) .. (5)

<223> X is independently Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (6) .. (6)

<223> X is independently from Y, G, S, R, A, D, E, F, H, I, K, L,

M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (6) .. (6)

<223> X is independently from Y, G, S, R, A, D, E, F, H, I, K, L,

M, N, P, Q, T, V, or W, and can be various lengths

<220>

<221> MISC_FEATURE <222> (7). . (7)

<223> X is independently from Y, G, S, R, A, D, E, F, H, I, K, L,

M, N, P, Q, T, V, or W

<220>

<221> MISC_FEATURE <222> (8) .. (8)

<223> X is independently from Y, G, S, R, A, D, E, F, H, I, K, L,

M, N, P, Q, T, V, or W <220>

<221> MISC_FEATURE <222> (9) .. (9)

<223> X is independently Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<400> 4

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Synthetic heavy chain articulation sequence <400> 5

Thr Cys Pro Pro Cys Pro Wing Pro Glu Read Leu Gly15 10

<210> 6

<211> 23

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5-8 LC-FR1 <400> 6

Asp lie Gln Met Thr Gln Ser Pro Ser Ser Ser Ala Ser Ser Gly1 5 10 15

Asp Arg Go Thr Lie Thr Cys20

<210> 7

<211> 15

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 LC-FR2 <400> 7

Trp Tyr Gln Gln Lys Pro Gly Lys Pro Wing Lys Leu Leu lie Tyr1 5 10 15

<210> 8 <211> 32 <212> PRT <213> Artificial <220>

<223> synthetic huMAb4D5 LC-FR3 <400> 8

Gly Goes To Be Arg Phe Be Gly Be Arg Be Gly Thr Asp Phe Thr1 5 10 15

Read Thr lie Ser Be Read Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys20 25 30

<210> 9

<211> 10

<212> PRT

<213> Artificial

<220>

<223> huMAb4D5 synthetic LC-FR4 <400> 9

Phe Gly Gln Gly Thr Lys Goes Glu lie Lys1 5 10

<210> 10

<211> 25

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5-8 HC-FR1 <400> 10

Glu Goes Gln Leu Goes Glu Be Gly Gly Gly Leu Goes Gln Pro Gly Gly1 5 10 15

Ser Leu Arg Le Ser Ser Cys Wing Ser20 25

<210> 11

<211> 13

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR2 <400> 11

Trp Going Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Going 1 5 10

<210> 12 <211> 30

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR3 <400> 12

Arg Phe Thr lie Be Wing Asp Thr Be Lys Asn Thr Wing Tyr Leu Gln1 5 10 15

Met Asn Be Read Arg Wing Glu Asp Thr Wing Go Tyr Tyr Cys20 25 30

<210> 13

<211> 11

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR4 <4u0> 13

Trp Gly Gln Gly Thr Leu Will Thr Will Be Ser1 5 10

<210> 14

<211> 23

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5-8 LC-FR1 <400> 14

Asp lie Gln Met Thr Gln Ser Pro Ser Ser Ser Ala Ser Ser Gly1 5 10 15

Asp Arg Go Thr Lie Thr Cys20

<210> 15 <211> 15 <212> PRT <213> Artificial

<400> 15

Trp Tyr Gln Gln Lys Pro Gly Lys Pro Wing Lys Leu Leu lie Tyr1 5 10 15

<210> 16 <211> 32 <212> PRT

<213> Artificial

<220>

<223> huMAb4D5 synthetic LC-FR3 <400> 16

Gly Gonna Be Arg Phe Be Gly Gly Be Gly Be Gly Thr Asp Phe Thr1 5 10 15

Read Thr lie Be Ser Read Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys20 25 30

<210> 17

<211> 10

<212> PRT

<213> Artificial

<220>

<223> huMAb4D5 synthetic LC-FR4 <400> 17

Phe Gly Gln Gly Thr Lys Goes Glu lie Lys1 5 10

<210> 18

<211> 25

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5-8 HC-FR1 <400> 18

Glu Goes Gln Leu Goes Glu Be Gly Gly Gly Leu Goes Gln Pro Gly Gly1 5 10 15

Ser Leu Arg Le Ser Ser Cys Wing Ser20 25

<210> 19

<211> 13

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR2 <400> 19

Trp Going Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Going 1 5 10 <210> 20

<211> 30

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR3

<400> 20

Arg Phe Thr lie Be Arg Asp Asn Be Lys Asn Thr Read Tyr Leu Gln1 5 10 15

Met Asn Be Read Arg Wing Glu Asp Thr Wing Go Tyr Tyr Cys20 25 30

<210> 21

<211> 11

<212> PRT

<213> Artificial

<220>

<223> synthetic huMAb4D5 HC-FR4

<400> 21

Trp Gly Gln Gly Thr Leu Will Thr Will Be Ser1 5 10

<210> 22

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (7). . (7)

<223> X is Y or S <22'0>

<221> MISC_FEATURE <222> (8) .. (8)

<223> X is Y or S

<400> 22

Gly Phe Xaa lie Xaa Xaa Xaa Xaa lie His1 5 10

<210> 23

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (8). . (8)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (10). . (10)

<223> X is Y or S

<400> 23

Xaa lie Xaa Pro Xaa Xaa Gly Xaa Thr Xaa Tyr Ala Asp Will Go Lys1 5 10 15

Gly

<210> 24 <211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (5). . (5)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is Y, G, or S

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (8) .. (8)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (9) .. (9)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (11) .. (11)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (12). . (12)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (13) .. (13)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (15) .. (15)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (17). . (17)

<223> X is Y, G, or S, or no amino acid <220>

<221> MISC_FEATURE

<222> (18) .. (18)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (19) .. (19)

<223> X is I, M, L, or F

<400> 24

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 25

<211> 9

<212> PRT <213> Artificial

<220>

<223> Synthetic CDRL3 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (8) .. (8)

<223> X is Y or S

<400> 25

Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1 5

<210> 26

<211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y, R, or S

<220>

<221> MISC_FEATURE

<222> (2). . (2)

<223> X is Y, R, or S

<220>

<221> MISC_FEATURE

<222> (3) .. (3) <table> table see original document page 278 </column> </row> <table> <220>

<221> MISC_FEATURE <222> (15) .. (15)

<223> X is Y, S, or R, or not present <220>

<221> MISC_FEATURE <222> (16) .. (16)

<223> X is Y, S, or R, or not present <220>

<221> MISC_FEATURE <222> (17) .. (17)

<223> X is Y, S, or R, or not present <220>

<221> MISC_FEATURE <222> (18) .. (18)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (19) .. (19)

<223> X is I, M, L, or F

<400> 26

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 27

<211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y, G, R, or S

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is Y, G, R, or S

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y, G, R, or S

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222><223><220><221><222><223><220><221><222> <223>

<220><221><222> <223>

MISC_FEATURE (4) .. (4)

X is Y, G, R, or S

MISC_FEATURE (5) .. (5)

X is Y, G, R, or S

MISC_FEATURE (6) .. (6)

X is Y, G, R, or S

MISC_FEATURE (7). . (7)

X is Y, S, G, R, or no amino acids

MISC_FEATURE (8) .. (8)

X is Y, S, G, R, or no amino acids

MISC_FEATURE (9) • • (9)

X is Y, S, G, R, or no amino acids

MISC_FEATURE (10) .. (10)

X is Y, S, G, R, or no amino acids

MISC_FEATURE (11) •• (11)

X is Y, S, G, R, or no amino acids

MISC_FEATURE

(12) .. (12)

X is Y, S, G, R, or no amino acids

MISC_FEATURE

(13) .. (13)

X is Y, S, G, R, or no amino acids

MISC_FEATURE

(14) .. (14)

X is Y, S, G, R, or no amino acids

MISC_FEATURE (15) .. (15)

X is Y, S, G, R, or no amino acid <220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is Y, S, G, R, or no amino acid <220>

<221> MISC_FEATURE

<222> (17) .. (17)

<223> X is Y, S, G, R, or no amino acid <220>

<221> MISC_FEATURE

<222> (18) .. (18)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (19) .. (19)

<223> X is I, M, L, or F

<400> 27

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa15 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 28

<211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE <222> (1) .. (1)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V,

or W

<220>

<221> MISC_FEATURE <222> (2) .. (2)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V,

or W

<220>

<221> MIS C_ FEAT URE <222> (3) .. (3)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, or W

<220>

<221> MISC FEATURE <table> table see original document page 18 </column> </row> <table> W, no amino acid

<220>

<221> MISC_FEATURE

<222> (14) .. (14)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, W, au no amino acid

<220>

<221> MISC_FEATURE

<222> (15) .. (15)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, W, au no amino acid

<220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, W, au no amino acid

<220>

<221> MISC_FEATURE

<222> (17). . (17)

<223> X is Y, G, S, R, A, D, E, F, H, I, K, L, M, N, P, Q, T, V, W, au no amino acid

<220>

<221> MISC_FEATURE

<222> (18) .. (18)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (19) .. (19)

<223> X is I, M, L, or F

<400> 28

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa15 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 29

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Synthetic Kabat Consensus CDRLl <400> 29

Arg Wing Be Gln Asp Go Asn Thr Wing Go Ala15 10 <210> 30

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Synthetic Kabat Consensus CDRL2

<400> 30

Ser Ala Ser Ser Leu Tyr Ser

<210> 31

<211> 18

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1). . (1)

<223> X is R, Y, or M

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is Y or R

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y, S, R, P, or G

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is Y, S, R, or H

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is R, Y, or S

<220>

<221> MISC_FEATURE

<222> (7). . (7)

<223> X is G, Y, or S <220>

<221> MISC_FEATURE

<222> (8). . (8)

<223> X is R, Y, or S

<220>

<221> MISC_FEATURE

<222> (9) .. (9)

<223> X is G, Y, or S

<220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is R, Y, or S

<220>

<221> MISC_FEATURE

<222> (11). . (11)

<223> X is G, Y, or S

<220>

<221> MISC_FEATURE

<222> (12). . (12)

<223> X is S, Y, R, G, or A

<220>

<221> MISC_FEATURE

<222> (13). . (13)

<223> X is G or Y

<220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is either L, M, R, G, or A <220>

<221> MISC_FEATURE

<222> (15). . (15)

<223> X is G, F, L, or no amino acid <220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is F or no amino acids

<400> 31

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15

Asp Tyr

<210> 32 <211> 45 <212> DNA <213> Artificial <220>

<223> Synthetic Hl

<400> 32

gcagcttctg gcttctmatt tmttmtmtmt atacactggg tgcgt 45

<210> 33

<211> 58

<212> DNA

<213> Artificial

<220>

<223> Synthetic H2

<400> 33

ctggaatggg ttgcatmtat ttmtccatmt tggttmtact tmttatgccg atagcgtc 58

<210> 34

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Synthetic L3

<400> 34

acttattact gtcagcaatm ttmttmttmt ccatmtacgt tcggacaggg tacc 54

<210> 35

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A6 <220>

<221> misc_feature

<222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG <220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 35

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnngstw tkgactactg gggtcaagga

<210> 36

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-A7 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC; TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33) <223> N31 through N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGG, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 36

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnng stwtkgacta ctggggtcaagga

<210> 37

<211> 66

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A8 <220>

<221> misc_feature

<222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature

<222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 37

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nngstwtkga ctactggggt

caagga

<210> 38

<211> 69

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A9

<220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG. AGT, AGC, GGA, GGT, GGC, or GGG <220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40). . (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43). . (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 38

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnngstwt kgactactgg

ggtcaagga

<210> 39

<211> 72

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A10

<220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220> <221>

misc feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N4 6 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 39

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngs twtkgactac

tggggtcaag ga

<210> 40

<211> 75

<212> DNA

<213> Artificial

<220> <223> Synthetic H3-A11 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220> <221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 40

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngstwtkgac

tactggggtc aagga

<210> 41

<211> 78

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A12 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 41

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngstwtk 60

gactactggg gtcaagga 78

<210> 42

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-A13 <220>

<221> misc_feature

<222> (19) .. (21) <223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG <220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 42

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngst

wtkgactact ggggtcaagg a

<210> 43

<211> 84

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A14

<220>

<221> misc_feature <222> (19). . (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220> <221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (58) .. (60) <223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGG, GGA, GGT, GGC, or GGG

<400> 43

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

gstwtkgact actggggtca agga 84

<210> 44

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-A15 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 44

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnngstwtkg actactgggg tcaagga 87

<210> 45 <211> 90

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-A16

<220>

<221> misc_feature <222> (19). . (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220> <221> misc_feature <222> (43). . (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (58). . (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 45

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn

nnnnnngstw tkgactactg gggtcaagga

<210> 46

<211> 93

<212> DNA

<213> Artificial <220>

<223> synthetic H3-A17 <220>

<221> misc_feature <222> (19). . (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently either TAT, TAC, TCA, TCT, TCC, TCG,

AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N4 9 through N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently either TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<220>

<221> misc_feature <222> (67) .. (69)

<223> N67 to N69 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, or GGG

<400> 46

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnng stwtkgacta ctggggtcaa gga 93 <210> 47 <211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B6 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 47

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnngstw tkgactactg gggtcaagga

<210> 48

<211> 63

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B7 <220>

<221> misc feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 48

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnng stwtkgacta ctggggtcaa 60

gga

<210> 49

<211> 66

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B8 <220>

<221> misc_feature

<222> (19) .. (21)

<223> N19 through N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28). . (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40). . (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 49

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nngstwtkga ctactggggt

caagga

<210> 50

<211> 69

<212> DNA

<213> Artificial

<220> <223> Synthetic H3-B9 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG <400> 50

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnngstwt kgactactgg 60

ggtcaagga 69

<210> 51

<211> 72

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B10 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (40). . (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 51

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngs twtkgactac

tggggtcaag ga

<210> 52

<211> 75

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B11 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221>

misc feature <222> (31). . (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N9 through N51 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 52

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngstwtkgac

tactggggtc aagga

<210> 53

<211> 78

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B12 <220>

<221> misc_feature

<222> (19) .. (21) <223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43). . (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG <220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 53

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngstwtk

gactactggg gtcaagga

<210> 54

<211> 81

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B13 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 54

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngst

wtkgactact ggggtcaagg a

<210> 55

<211> 84

<212> DNA

<213> Artificial <220>

<223> synthetic H3-B14 synthetic <220> <221> misc_feature <222> (19) .. (21) <223> N19 to N21 are independently TAT, TAC, TCA, TCT, CCT, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (22) .. (24) <223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (25) .. (27) <223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (28) .. (30) <223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (31) .. (33) <223> N31 through N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (34) .. (36) <223> N34 through N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <22l> misc_feature <222> (37) .. (39) <223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (40) .. (42) <223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, OR AGG

<220> <221> misc_feature <222> (43) .. (45) <223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCCTCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 55

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn

gstwtkgact actggggtca agga

<210> 56

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-B15 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG <220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N9 through N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221>

misc feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 56

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnngstwtkg actactgggg tcaagga 87

<210> 57

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-B16 <220>

<221> misc_feature

<222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (28) .. (30) <223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG <220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 57

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnnnnngstw tkgactactg gggtcaagga 90

<210> 58

<211> 93

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-B17 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 through N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> -----------------------------------......------ ---------------------- - -......... —------------ ^ --- ---------

<221> misc_feature <222> (43). . (45)

<223> N43 through N45 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60) <223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (67) .. (69)

<223> N67 to N69 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 58

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnnnnnnnng stwtkgacta ctggggtcaa gga 93

<210> 59

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C6 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (28). . (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 59

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnngstw tkgactactg gggtcaagga 60

<210> 60 '-: * • *

<211> 63

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C7 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31). . (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 60

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnng stwtkgacta ctggggtcaa 60

gga 63

<210> 61

<211> 66

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C8 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, GGA, GGT, GGC, GGG, AGT, AGC, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 61

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nngstwtkga ctactggggt 60

caagga

<210> 62

<211> 69

<212> DNA

<213> Artificial

66

<220>

<223> Synthetic H3-C9

<220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG <220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (31). . (33) ~~ ■ "~ '*" "

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA,

or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG <400> 62

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnngstwt kgactactgg 60

ggtcaagga 69

<210> 63

<211> 72

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C10 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N22 to N24 are independently ^ TÀT, "TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31). . (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43). . (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (46) .. (48)

<223> N4 6 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA,

or AGG

<400> 63

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngs twtkgactac 60

tggggtcaag ga 72

<210> 64

<211> 75

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C11 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221>

misc feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (28). . (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<23> _N34 to N36 are independently TAT, TAC, TCA, TCTJTOÇ, _TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220> <221>

misc feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<400> 64

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngstwtkgac 60

tactggggtc aagga 75

<210> 65

<211> 78

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C12 <220>

<221> misc_feature

<222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA,

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (28) .. (30) ___

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36) <223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA,

~ ™ or 'AGG' '"~" "'

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA, or AGG

<220>

<221> misc_feature

<222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG, AGA,

or AGG

<400> 65

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngstwtk 60

gactactggg gtcaagga 78

<210> 66

<211> 81

<212> DNA

<213> Artificial

<220> <223> Synthetic H3-C13 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGAor AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGAor AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, -AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA ^ or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AG A, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N4 9 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<400> 66

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnn nnnnnnngst 60wtkgactact ggggtcaagg a 81

<210> 67

<211> 84

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C14 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG <220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CG A, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature

<222> (31). . (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG <220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AG A, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N4 9 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<400> 67

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

gstwtkgact actggggtca agga 84

<210> 68

<211> 87

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C15 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220> misc_feature (22). . (24)

N22 to N24 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, GGG, CG A, CGT, CGC, CGG. AGA, or AGG

misc_feature (25) .. (27)

N25 to N27 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

mi sc_feature (28) .. (30)

N28 to N30 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

misc_feature

(31) .._ (33_) <223> N31 to N33 are independently TAT, "TAC," TCA, "TCT," TCC, "

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (40). . (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (43). . (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221><222> <223>

<220><221><222> <223>

<220><221><222> <223>

<220><221><222> <221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature

<222> (55) .. (57)

<223> N55 - N57 ~ "areindependent" "TAT, TAC, TCA," "TCT," TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA,

or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<400> 68

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnngstwtkg actactgggg tcaagga 87

<210> 69

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C16

<220>

<221> misc feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGAor AGG

<220>

<221> misc_feature <222> (22). . (24)

<223> N22 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGAor AGG

<220>

<221> misc_feature <222> (25). . (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28_ through N30 are independently TAT, TAC, TCA, TCT, _TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (31) .. (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (46). . (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (52) .. (54)

<223> N52 to N5j4 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> N55 to N57 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature

<222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<400> 69

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnngstw tkgactactg gggtcaagga 9

<210> 70

<211> 93

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C17 <220>

<221> misc_feature <222> (19) .. (21)

<223> N19 to N21 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AG A, or AGG

<220>

<221> misc_feature <222> (22) .. (24)

<223> N21 to N24 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (25) .. (27)

<223> N25 to N27 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (28) .. (30)

<223> N28 to N30 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (31). . (33)

<223> N31 to N33 are independently TAT, TAC, TCA, TCT, TCC, _______

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (34) .. (36)

<223> N34 to N36 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (37) .. (39)

<223> N37 to N39 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, GGG, CG A, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (40) .. (42)

<223> N40 to N42 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (43) .. (45)

<223> N43 to N45 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (46) .. (48)

<223> N46 to N48 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (49) .. (51)

<223> N49 to N51 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (52). . (54)

<223> N52 to N54 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (55) .. (57)

<223> _N55._ to N57 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG-AGA, or AGG

<220>

<221> misc_feature <222> (58) .. (60)

<223> N58 to N60 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (61) .. (63)

<223> N61 to N63 are independently TAT, TAC, TCA, TCT, TCC, TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (64) .. (66)

<223> N64 to N66 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<220>

<221> misc_feature <222> (67) .. (69)

<223> N67 to N69 are independently TAT, TAC, TCA, TCT, TCC,

TCG, AGT, AGC, GGA, GGT, GGC, GGG, CGA, CGT, CGC, CGG. AGA, or AGG

<400> 70

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnnnnnnnng stwtkgacta ctggggtcaa gga 93

<210> 71

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C6 <220>

<221> misc_feature

<222> (19) .. (36)

<223> N19 through N36 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 71

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnngstw tkgactactg gggtcaagga 60

<210> 72

<211> 63

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C7 <220>

<221> misc_feature

<222> (19) .. (39)

<223> N19 to N39 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG

<400> 72

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnng stwtkgacta ctggggtcaagga 63

<210> 73

<211> 66

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C8 <220>

<221> misc_feature

<222> (19) .. (42)

<223> N19 to N42 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG

<400> 73

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nngstwtkga ctactggggt 60

caagga 66

<210> 74

<211> 69

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C9

<220>

<221> misc_feature <222> (19) .. (45)

<223> N19 through N45 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 74

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnngstwt kgactactgg 60

ggtcaagga 69

<210> 75

<211> 72

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C10 <220>

<221> misc_feature

<222> (19) .. (48)

<223> N19 to N48 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG

<400> 75

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngs twtkgactac 60tggggtcaag ga 72

<210> 76

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-C11 <220>

<221> misc_feature

<222> (19) .. (51)

<223> N19 through N51 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 76

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngstwtkgac 60tactggggtc aagga 75 <210> 77

<211> 78

<212> "DNA

<213> Artificial

<220>

<223> Synthetic H3-C12

<220>

<221> misc_feature <222> (19) .. (54)

<223> N19 to N54 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 77

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngstwtk

gactactggg gtcaagga

<210> 78

<211> 81

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C13

<220>

<221> misc_feature <222> (19) .. (57)

<223> N19 to N57 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG

<400>

78gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngst 60

wtkgactact ggggtcaagg a 81

<210> 79

<211> 84

<212> DNA

<213> Artificial

<220>

<223> Synthetic H3-C14 <220>

<221> misc_feature

<222> (19) .. (60)

<223> N19 to N60 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG

<400> 79

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60gstwtkgact actggggtca agga 84

<210> 80

<211> 87

<212> DNA

<213> Artificial

<220> -----------------—

<223> Synthetic H3-C15

<220>

<221> misc_feature <222> (19) .. (63)

<223> N19 to N63 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 80

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnngstwtkg actactgggg tcaagga 87

<210> 81

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic H3-C16 <220>

<221> misc_feature

<222> (19) .. (66)

<223> N19 to N66 is any combination of A, T, C, or G except TGT, TGC, TGA, TAA, TAG <400> 81

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

nnnnnngstw tkgactactg gggtcaagga

<210> 82

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> Synthetic H3-C17 <220>

<221> misc_feature

<222> (19) .. (69)

<223> N19 to N69 is any combination of A, T, C, or G, exceptTGT, TGC, TGA, TAA, TAG

<400> 82

gtctattatt gtgctcgcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnn 60

. nnnnnnnnng stwtkgacta ctg.gggtcaa gga ........._ 93

<210> 83

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 1CDRL3 synthetic HER2 ligand <400> 83

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 84

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 3CDRL3 ligand

<400> 84

Gln Gln Be Ser Tyr Tyr Pro Ser Thr1 5

<210> 85

<211> 9

<212> PRT

<213> Artificial

<220> <223> Artificial HER2 binder synthetic clone 3CDRL3

<400> 85

Gln Gln Be Ser Tyr Tyr Pro Ser Thr1 5

<210> 86

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 4CDRL3 binder

<400> 86

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 87

<211> 9

<212> PRT

<213> Artificial "<220>

<223> Artificial synthetic clone 5CDRL3 HER2 ligand

<400> 87

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 88

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 6CDRL3 binder

<400> 88

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 89

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 7CDRL3 binder

<400> 89

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5 <210> 90

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 8CDRL3 binder

<400> 90

Gln Tyr Tyr Tyr Tyr Pro Tyr Thr1 5

<210> 91

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 9CDRL3 synthetic HER2 ligand

<400> 91

Gn "Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 92

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 10CDRL3 synthetic HER2 ligand

<400> 92

Gln Gln Being Tyr Tyr Tyr Pro Tyr Thr1 5

<210> 93

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 11CDRL3 synthetic HER2 ligand

<400> 93

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 94

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial clone 12CDRL3 synthetic HER2 ligand

<400> 94

Gln Gln His Tyr Thr Thr Pro Pro Thr1 5

<210> 95

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 13CDRL3 binder

<400> 95

Gln Gln Tyr Tyr Tyr Pro Tyr Thr

<210> 96

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 14CDRL3 synthetic HER2 ligand

<400> 96

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 97

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 15CDRL3 synthetic HER2 ligand

<400> 97

Gln Gln Tyr Tyr Tyr Pro Tyr Thr

<210> 98

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 16CDRL3 synthetic HER2 ligand

<400>

98Gln Gln Being Ser Tyr Being Pro Ser Thr1 5

<210> 99

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 17CDRL3 synthetic HER2 ligand

<400> 99

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 100

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 18CDRL3 synthetic HER2 ligand

<400> 100

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 101

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 19CDRL3 synthetic HER2 binder

<400> 101

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 102

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 20CDRL3 binder

<400> 102

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 103 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 21CDRL3

<400> 103

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 104

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 22CDRL3

<400> 104

Gln Gln Tyr Tyr Tyr Pro Tyr Thr

15 ~ "

<210> 105

<211> 9

<212> PRT

<213> Artificial

<220>

<223> A-rtrf iclal-Tclane- "23CDRL-3-synthetic HER2 binder

<400> 105

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 106

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 24CDRL3 HER2 ligand

<400> 106

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 107

<211> 9

<212> PRT

<213> Artificial

<220> <223> Artificial HER2 binder synthetic clone 25CDRL3

<400> 107

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 108

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 26CDRL3 synthetic HER2 ligand

<400> 108

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 109

<211> 9

<212> PRT <213> Artificial

<220>

<223> Artificial clone 27CDRL3 synthetic HER2 ligand

<400> 109

Gln Gln Tyr Tyr Tyr Pro Tyr Thr

1 5

<210> 110

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 28CDRL3 binder

<400> 110

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 111

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone 29CDRL3 synthetic ligand

<400> 111

Gln Gln Be Ser Tyr Ser Pro Ser Thr1 5 <210> 112

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 30CDRL3 HER2 synthetic binder

<400> 112

Gln Gln Being Ser Tyr Tyr Pro Tyr Thr

<210> 113

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 31CDRL3 binder

<400> 113

Gln Gln "Ser Tyr Tyr Tyr ProSer Thr

<210> 114

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 32CDRL3 binder

<400> 114

Gln Gln Being Ser Tyr Tyr Pro Tyr Thr

<210> 115

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 33CDRL3 binder

<400> 115

Gln Gln Be Phe Be Ser Pro Tyr Thr1 5

<210> 116

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial HER2 synthetic clone 34CDRL3 binder

<400> 116

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 117

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 35CDRL3 binder

<400> 117

Gln Gln Ser Tyr Ser Tyr Pro Ser Thr1 5

<210> 118

<211> 9 <212> "PRT

<213> Artificial

<220>

<223> Artificial HER2 binder synthetic clone 36CDRL3

<400> 118

Gln Gln Be Phe Ser Tyr Pro Tyr Thr "1 5

<210> 119

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 clone 37CDRL3 binder

<400> 119

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 120

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder 38CDRL3 clone

<400> 120Gln Gln Ser Tyr Ser Ser Pro Ser Thr1 5

<210> 121

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 39CDRL3 synthetic HER2 ligand

<400> 121

Gln Gln Ser Tyr Ser Tyr Pro Ser Thr1 5

<210> 122

<211> 9

<212> PRT

<213> Artificial

<220>

<22.3>. Artificial HER2 clone 40CDRL3 synthetic binder

<400> 122

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 123

<211> 9

<212> PRT ~ -

<213> Artificial

<220>

<223> Artificial synthetic HER2 clone 41CDRL3 binder

<400> 123

Gln Gln Ser Tyr Ser Ser Ser Pro 5

<210> 124

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 42CDRL3 binder

<400> 124

Gln Gln Ser Tyr Ser Ser Ser Pro 5

<210> 125 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 43CDRL3 binder

<400> 125

Gln Gln Ser Phe Ser Ser Ser Pro 5

<210> 126

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 44CDRL3 binder

<400> 126

^ Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr

<210> 127

<211> 9

<212> PRT

<213> Artificial

<220>

<223> ArtChapter 45CDRL3 synthetic binder give HER2

<400> 127

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 128

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 46CDRL3

<400> 128

Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 129

<211> 9

<212> PRT

<213> Artificial

<220> <223> Artificial clone 4 7CDRL3 synthetic HER2 ligand

<400> 129

Gln Gln Ser Tyr Ser Ser 1 5 Pro Ser Thr

<210> <211> <212> <213> 130 9 Artificial PRT

<220> <223> Artificial HER2 binder synthetic clone 48CDRL3

<400> 130

Gln Gln Ser Ser Ser Tyr 1 5 Pro Tyr Thr

<210> <211> <212> 131 9 PRT

<213> "Artificial"

<220> <223> Artificial clone 4 9CDRL3 synthetic HER2 ligand

<400> 131

Gln Gln Ser Tyr Ser Tyr 1 5 'Pro Ser Thr .... ......

<210> <211> <212> <213> 132 9 Artificial PRT

<220> <223> Artificial HER2 binder synthetic clone 50CDRL3

<400> 132

Gln Gln Ser Ser Ser Ser 1 1 Pro Tyr Thr

<210> <211> <212> <213> 133 9 Artificial PRT

<220> <223> Artificial HER2 binder synthetic clone 51CDRL3

<400> 133

Gln Gln Ser Ser Ser Ser Tyr Pro Tyr Thr1 5 <210> 134

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 52CDRL3 HER2 ligand

<400> 134

Gln Gln Ser Ser Ser Tyr Pro Ser Thr1 5

<210> 135

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 53CDRL3 HER2 ligand

<400> 135

Gln Gln Ser Tyr Ser Ser Ser Pro "1 5

<210> 136

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone synthetic binder 54CDRL3

<400> 136

Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 137

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 55CDRL3 binder

<400> 137

Gln Gln Ser Ser Ser Tyr Pro Ser Thr1 5

<210> 138

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial HER2 synthetic clone 56CDRL3 binder

<400> 138

Gln Gln Be Ser Tyr Tyr Pro Ser Thr1 5

<210> 139

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone 57CDRL3 synthetic ligand

<400> 139

Gln Gln Be Ser Phe Ser Pro Tyr Thr1 5

<210> 140

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 58CDRL3 binder

<400> 140

Gln Gln Ser Ser Ser Tyr Pro Tyr Thr1 5

<210> 141

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 59CDRL3 binder

<400> 141

Gln Gln Ser Ser Ser Tyr Pro Ser Thr1 5

<210> 142

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone 60CDRL3 synthetic binder

<400> 142Gln Gln Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 143

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 61CDRL3 synthetic HER2 ligand <400> 143

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 144

<211> 9

<212> PRT

<213> Artificial

<220>

<2-2-3> —A-r-ti-fig-1-clone — 6.2-CDRL3-will bind. Synthetic HER2 <400> 144

Gln Gln Ser Tyr Ser Ser Ser Pro 5

<210> 145

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 63CDRL3 synthetic HER2 ligand <400> 145

Gln Gln Ser Tyr Ser Ser Ser Pro 5

<210> 146

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 64CDRL3 HER2 ligand <400> 146

Gln Gln Ser Ser Ser Ser Tyr Pro Tyr Thr1 5

<210> 147 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 65CDRL3 binder

<400> 147

Gln Gln Be Phe Be Ser Pro Tyr Thr1 5

<210> 148

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 66CDRL3 binder

<400> 148

Gln Gln Ser Tyr Ser Tyr Pro Ser Thr

1 5

<210> 149

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 clone 67CDRL3 binder

<400> 149

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr1 5

<210> 150

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 clone 68CDRL3 binder

<400> 150

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 151

<211> 9

<212> PRT

<213> Artificial

<220> <223> Artificial HER2 binder synthetic clone 69CDRL3

<400> 151

Gln Gln Ser Ser Ser Tyr Pro Ser Thr1 5

<210> 152

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 70CDRL3 binder

<400> 152

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 153

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 71CDRL3 binder

<400> 153

Gln Gln Be Ser Tyr Tyr Pro Ser Thr1 5

<210> 154

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 72CDRL3 binder

<400> 154

Gln Gln Ser Be Phe Tyr Pro Ser Thr1 5

<210> 155

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 73CDRL3 HER2 synthetic binder

<400> 155

Gln Gln Ser Ser Tyr Ser Ser Tyr Thr1 5 <210> 156

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 74CDRL3

<400> 156

Gln Gln Ser Ser Ser Tyr Pro Ser Thr1 5

<210> 157

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 75CDRL3 binder

<400> 157

Gln Gln Ser Ser Ser Ser Ser Pro 5

<210> 158

<211> 9 ^

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 76CDRL3 binder

<400> 158

Gln Tyr Tyr Tyr Tyr Pro Tyr Thr1 5

<210> 159

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 77CDRL3 binder

<400> 159

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 160

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial HER2 synthetic binder clone 78CDRL3

<400> 160

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 161

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 79CDRL3 ligand

<400> 161

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 162

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 80CDRL3 binder

<400> 162

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 163

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 81CDRL3 synthetic HER2 ligand

<400> 163

Gln Gln Ser Tyr Ser Tyr Pro Ser Thr1 5

<210> 164

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone binder 82CDRL3

<400> 164Gln Gln Tyr Tyr Tyr Pro Ser Thr1 5

<210> 165

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 83CDRL3 binder

<400> 165

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 166

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 84CDRL3

<400> 166

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 167

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone binder 85CDRL3

<400> 167

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 168

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 86CDRL3 binder

<400> 168

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 169 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 87CDRL3 binder

<400> 169

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 170

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 88CDRL3 binder

<400> 170

Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 171

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 89CDRL3 binder

<400> 171

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 172

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 90CDRL3 binder

<400> 172

Gln Gln Be Ser Tyr Ser Pro Tyr Thr1 5

<210> 173

<211> 9

<212> PRT

<213> Artificial

<220> <223> Artificial HER2 binder synthetic clone 91CDRL3

<400> 173

Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 174

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 92CDRL3 binder

<400> 174

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 175

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone 93CDRL3 synthetic binder

<400> 175

Gln Gln Ser Ser Ser Ser Ser Pro Tyr Thr1 5

<210> 176

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone synthetic binder 94CDRL3

<400> 176

Gln Gln Being Ser Tyr Tyr Pro Tyr Thr1 5

<210> 177

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 95CDRL3 binder

<400> 177

Gln Gln Ser Tyr Tyr Tyr Pro Tyr Thr1 5 <210> 178

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 96CDRL3 ligand

<400> 178

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 179

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 97CDRL3 binder

<400> 179

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 180

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 98CDRL3 binder

<400> 180

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 181

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone binder 99CDRL3

<400> 181

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 182

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial HER2 synthetic clone binder 100CDRL3

<400> 182

Gln Gln Being Ser Tyr Tyr Pro Tyr Thr1 5

<210> 183

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone binder 101CDRL3

<400> 183

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 184

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 102CDRL3 binder

<400> 184

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 185

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 103CDRL3 HER2 ligand

<400> 185

Gln Gln Tyr Ser Tyr Tyr Pro Ser Thr1 5

<210> 186

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 104CDRL3 binder

<400>

186Gln Gln Tyr Ser Ser Ser Pro Ser Thr1 5

<210> 187

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 105CDRL3 HER2 ligand <400> 187

Gln Gln Tyr Ser Ser Ser Ser Tyr Thr1 5

<210> 188

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 106CDRL3 synthetic HER2 ligand <400> 188

Gln Gln Ser Tyr Ser Ser Ser Pro 5

<210> 189

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 1CDRL3 synthetic HER2 ligand <400> 189

Gly Phe Ser Lie Ser Ser Ser Tyr lie His15 10

<210> 190

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 2CDRL3 synthetic HER2 ligand <400> 190

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 191 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 3CDRL3 synthetic HER2 ligand <400> 191

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 192

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 4CDRL3 synthetic HER2 ligand <400> 192

Gly Phe Tyr lie. To be . Tyr Ser Tyr lie His1 5 10

<210> 193

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 5CDRL3 synthetic HER2 ligand <400> 193

Gly Phe Tyr lie Ser Ser Ser Tyr lie His15 10

<210> 194

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 6CDRL3 synthetic HER2 ligand <400> 194

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His 1 5 10

<210> 195

<211> 10

<212> PRT

<213> Artificial <220>

<223> Artificial clone 7CDRL3 synthetic HER2 ligand <400> 195

Gly Phe Ser Tyr Tyr Tyr Ser Tyr lie His15 10

<210> 196

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 8CDRL3 synthetic HER2 ligand <400> 196

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 197

<211> 10. __________________________........_______________ ............................ - ------

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 9CDRL3 synthetic HER2 ligand <400> 197

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 198

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 10CDRL3 synthetic HER2 ligand <400> 198

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 199

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 11CDRL3 synthetic HER2 ligand

<400> 199Gly Phe Ser Ile Tyr Ser Ser Ile His15 10

<210> 200

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 12CDRL3 synthetic HER2 ligand <400> 200

Gly Phe Being Ile Being Being Tyr Being Ile His15 10

<210> 201

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 13CDRL3 synthetic HER2 ligand. <400> 201

Gly Phe Ser Ile Ser Ser Ser Ser Ile His15 10

<210> 202

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 14CDRL3 HER2 ligand <400> 202

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 203

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 15CDRL3 synthetic HER2 ligand <400> 203

Gly Phe Ser Ile Ser Ser Ser Ser Ile His15 10

<210> 204 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 16CDRL3 synthetic HER2 ligand <400> 204

Gly Phe Tyr lie Be Ser Tyr Ser Lie His1 5 10

<210> 205

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 17CDRL3 synthetic HER2 ligand <400> 205

Gly Phe Tyr lie Ser Tyr Ser Ser Lie His1 5 10

<210> 206

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 18CDRL3 synthetic HER2 ligand <400> 206

Gly Phe Ser lie Ser Tyr Ser Ser Lie His1 5 10

<210> 207

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 19CDRL3 HER2 ligand <400> 207

Gly Phe Tyr lie Be Ser Tyr Ser Lie His1 5 10

<210> 208

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 20CDRL3 HER2 ligand <400> 208

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 209

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 21CDRL3 HER2 ligand <400> 209

Gly Phe Tyr lie Ser Tyr Ser Ser His15 10

<210> 210

<211> 10

<212> PRT. ..

<213> Artificial

<220>

<223> Artificial clone 22CDRL3 synthetic HER2 ligand <400> 210

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 211

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 23CDRL3 synthetic HER2 ligand <400> 211

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 212

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 24CDRL3 synthetic HER2 ligand <400> 212Gly Phe Ser ser Ser Tyr Ser ser His 5 5 10

<210> 213

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 25CDRL3 synthetic HER2 ligand <400> 213

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 214

<211> 10

<212> PRT

<213> Artificial

<220>

- <223> Ar-artificial-clone-2-CDRL3 -... binder. of synthetic HER2_

<400> 214

Gly Phe Tyr lie Ser Tyr Ser Ser His15 10

<210> 215

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 27CDRL3 synthetic HER2 ligand <400> 215

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 216

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 28CDRL3 HER2 ligand <400> 216

Gly Phe Ser Lie Ser Tyr Ser Lie His15 10

<210> 217 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 29CDRL3 synthetic HER2 ligand <400> 217

Gly Phe Ser Lie Tyr Ser Ser Lie His1 5 10

<210> 218

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 30CDRL3 synthetic HER2 ligand <400> 218

Gly Phe Ser Lie Ser Tyr Ser Lie Lie His

1 5 10

<210> 219

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 31CDRL3 synthetic HER2 ligand <400> 219

Gly Phe Ser lie Ser Tyr Ser Ser Lie His1 5 10

<210> 220

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 32CDRL3 synthetic HER2 ligand <400> 220

Gly Phe Ser Lie Ser Ser Ser Ser Ser His 5 5 10

<210> 221

<211> 10

<212> PRT

<213> Artificial

<220> <223> synthetic HER2 binding clone 33 CDRH1

<400> 221

Gly Phe Being Ile Being Being Tyr Tyr Ile His1 5 10

<210> 222

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 34CDRL3 binder

<400> 222

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 223

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 35CDRL3 binder

<400> 223

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 224

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 36CDRL3 synthetic HER2 ligand <400> 224

Gly Phe Being Ile Being Being Being Being Ile His1 5 10

<210> 225

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 37CDRL3 synthetic HER2 ligand <400> 225Gly Phe Tyr Ile Ser Ser Tyr Ser Ile His1 5 10

<210> 226

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 38CDRL3 synthetic HER2 ligand <400> 226

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 227

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Ariificial HER2 ligand clone 39CDRL3. synthetic

<400> 227

Gly Phe To Be Ile To Be To Be To Be Ile His1 5 10 <210> 228

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 40CDRL3 synthetic HER2 ligand <400> 228

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 229

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 41CDRL3 synthetic HER2 ligand <400> 229

Gly Phe Being Ile Being Being Being Being Ile His1 5 10

<210> 230 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 42CDRL3 synthetic HER2 ligand <400> 230

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 231

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 43CDRL3 synthetic HER2 ligand <400> 231

Gly Phe Ser lie Ser Ser Ser Ser Tle His15 10

<210> 232

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 44CDRL3 synthetic HER2 ligand <400> 232

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 233

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 45CDRL3 synthetic HER2 ligand <400> 233

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 234

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic HER2 binder clone 46CDRL3

<400> 234

Gly Phe Tyr lie Be Ser Tyr Ser Lie His1 5 10

<210> 235

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 47CDRL3 synthetic HER2 ligand <400> 235

Gly Phe Ser lie Ser Ser Tyr Ser Lie His1 5 10

<210> 236

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 48CDRL3 synthetic HER2 ligand <400> 236

Gly Phe Tyr lie Be Ser Tyr Tyr lie His1 5 10

<210> 237

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 49CDRL3 HER2 ligand <400> 237

Gly Phe Ser lie Ser Ser Tyr Ser Lie His1 5 10

<210> 238

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 50CDRL3 synthetic HER2 ligand <400> 238Gly Phe Tyr Ile Ser Ser Ser Ile His1 5 10

<210> 239

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 51CDRL3 synthetic HER2 ligand <400> 239

Gly Phe Being Ile Being Being Being Being Ile His1 5 • 10

<210> 240

<211> 10

<212> PRT

<213> Artificial

<220>

<-2-2-3> —A-thi-FHL-eia-1-β-ne--2-G4DR-L3-, 1-H-ER2-synthetic Î ± -400 <400> 240

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 241

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 53CDRL3 synthetic HER2 ligand <400> 241

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 242

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 54CDRL3 synthetic HER2 ligand <400> 242

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 243 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 55CDRL3 binder

<400> 243

Gly Phe Ser Lie Ser Ser Ser Ser Ser His 5 5 10

<210> 244

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 56CDRL3 synthetic HER2 ligand <400> 244

Gly Phe Ser IIe Ser Ser Ser Ser Ile His

1 5 10

<210> 245

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 57CDRL3 synthetic HER2 ligand <400> 245

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 246

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 58CDRL3 synthetic HER2 ligand <400> 246

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 247

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial clone 59CDRL3 synthetic HER2 ligand <400> 247

Gly Phe Tyr lie Be Ser Tyr Ser Lie His1 5 10

<210> 248

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 60DRL3 synthetic HER2 ligand <400> 248

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 10

<210> 249 <211> 10

<2l2-> - PRT <2l3> "Artificial"

<220>

<223> Artificial clone 61CDRL3 synthetic HER2 ligand <400> 249

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 10

<210> 250

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 62CDRL3 synthetic HER2 ligand <400> 250

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 10

<210> 251

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic binder clone 63CDRL3

<400> 251Gly Phe Tyr Ile Ser Ser Ser Ser Ile His15 10

<210> 252

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 64CDRL3 synthetic HER2 ligand <400> 252

Gly Phe Tyr Ile Be Ser Tyr Be Ile His1 5 10

<210> 253

<211> 10

<212> PRT

<213> Artificial

<220>

<223J> _Artificial

<400> 253

Gly Phe Tyr Ile Be Ser Be Tyr Ile His15 10

<210> 254

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 66CDRL3 synthetic HER2 ligand <400> 254

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 255

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 67CDRL3 synthetic HER2 ligand <400> 255

Gly Phe Tyr Ile Be Tyr Be Ile His15 10

<210> 256 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 68CDRL3 synthetic HER2 ligand <400> 256

Gly Phe Ser lie Tyr Tyr Ser Lie His15 10

<210> 257

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 69CDRL3 synthetic HER2 ligand <400> 257

1 510

<210> 258

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 70CDRL3 synthetic HER2 ligand <400> 258

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 10

<210> 259

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 71CDRL3 synthetic HER2 ligand <400> 259

Gly Phe Tyr lie Ser Ser Tyr Ser Ser His15 10

<210> 260

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial clone 72CDRL3 synthetic HER2 ligand <400> 260

Gly Phe Tyr lie Ser Tyr Tyr Ser lie His1 5 10

<210> 261

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 73CDRL3 synthetic HER2 ligand <400> 261

Gly Phe Tyr lie Ser Tyr Tyr Ser lie His1 5 10

<210> 262 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 74CDRL3 synthetic HER2 ligand <400> 262

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 263

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 75CDRL3 synthetic HER2 ligand <400> 263

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 264

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 76CDRL3 binder

<400> 264Gly Phe Tyr Ile Ser Ser Ser Ser Ile His1 5 10

<210> 265

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 77CDRL3 synthetic HER2 ligand <400> 265

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 266

<211> 10

<212> PRT

<213> Artificial

<220>

<400> 266

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 267

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 79CDRL3 synthetic HER2 ligand <400> 267

Gly Phe Ser Ile Ser Ser Ser Ser Ile His15 10

<210> 268

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 80CDRL3 synthetic HER2 ligand <400> 268

Gly Phe Being Ile Tyr Tyr Being Ile His15 10

<210> 269 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 81CDRL3 synthetic HER2 ligand <400> 269

Gly Phe Tyr lie Ser Ser Ser Tyr lie His1 5 10

<210> 270

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 82CDRL3 synthetic HER2 ligand <400> 270

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 271

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 83CDRL3 synthetic HER2 ligand <400> 271

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 272

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 84CDRL3 <400> 272

Gly Phe Ser Lie Ser Ser Ser Ser Ser His 5 5 10

<210> 273

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic HER2 binder clone 85CDRL3 <400> 273

Gly Phe Ser Lie Ser Ser Ser Ser Ser His 5 5 10

<210> 274

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 86CDRL3 synthetic HER2 ligand <400> 274

Gly Phe Ser lie Ser Tyr Ser Ser Lie His1 5 10

<210> 275

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 87CDRL3 synthetic HER2 ligand <400> 275

Gly Phe Tyr lie Ser Tyr Ser Ser Lie His1 5 10

<210> 276

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 88CDRL3 synthetic HER2 ligand <400> 276

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 277

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 89CDRL3 synthetic HER2 ligand <400> 277Gly Phe Ser Ile Ser Ser Ser Ile His1 5 10

<210> 278

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 90CDRL3 synthetic HER2 ligand <400> 278

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 279

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 91CDRL3 synthetic HER2 ligand <400> 279

Gly Phe Being Ile Being Being Being Being Ile His1 5 10

<210> 280

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 92CDRL3 synthetic HER2 ligand <400> 280

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 281

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 93CDRL3 synthetic HER2 ligand <400> 281

Gly Phe Tyr Ile Being Being Being Being Ile His1 5 10

<210> 282 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone synthetic binder 94CDRL3

<400> 282

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 283

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 95CDRL3 synthetic HER2 ligand <400> 283

Gly Phe Phe lie Ser Tyr Tyr Tyr lie His1 5 10

<210> 284

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder 96CDRL3 clone <400> 284

Gly Phe Ser lie Tyr Ser Ser Tyr lie His1 5 10

<210> 285

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 97CDRL3 synthetic HER2 ligand <400> 285

Gly Phe Tyr lie Ser Tyr Ser Ser Lie His1 5 10

<210> 286

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic HER2 ligand clone 98CDRL3 <400> 286

Gly Phe Tyr lie Tyr Ser Tyr Ser Lie His1 5 10

<210> 287

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 99CDRL3 synthetic HER2 ligand <400> 287

Gly Phe Tyr lie Ser Tyr Ser Ser Lie His1 5 10

<210> 288

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 100CDRL3 HER2 ligand <400> 288

Gly Phe Ser lie Ser Tyr Ser Ser Lie His1 5 10

<210> 289

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 101CDRL3 synthetic HER2 ligand <400> 289

Gly Phe Ser lie Ser Tyr Ser Ser Lie His1 5 10

<210> 290

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 102CDRL3 synthetic HER2 ligand <400> 290Gly Phe Tyr Ile Ser Tyr Ser Ile His1 5 10

<210> 291

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 103CDRL3 synthetic HER2 ligand <400> 291

Gly Phe Ser Ile Ser Tyr Ser Ser Ile His1 5 10

<210> 292

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 104CDRL3 synthetic HER2 ligand <400> 292

Gly Phe Ser Ile Ser Tyr Ser Tyr Ile His1 5 10

<210> 293

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 105CDRL3 synthetic HER2 ligand <400> 293

Gly Phe Ser Ile Ser Tyr Ser Tyr Ile His1 5 10

<210> 294

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 106CDRL3 synthetic HER2 ligand <400> 294

Gly Phe Tyr Ile Be Tyr Be Tyr Ile His1 5 10

<210> 295 <211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 1CDHR2 synthetic HER2 ligand <400> 295

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 296

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 2CDHR2 synthetic HER2 ligand <400> 296

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 297

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 3CDHR2 synthetic HER2 ligand <400> 297

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 298

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 4CDHR2 synthetic HER2 ligand <400> 298

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15Gly

<210> 299

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 5CDHR2 HER2 ligand

<400> 299

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 300

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 6CDHR2 HER2 synthetic binder

<400> 300

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 301

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 7CDHR2 synthetic HER2 ligand

<400> 301

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 302

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 8CDHR2

<400> 302Be lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 303

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 9CDHR2 synthetic HER2 ligand <400> 303

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 304

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 10CDHR2 synthetic HER2 ligand <400> 304

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 305

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 11CDHR2 synthetic HER2 ligand <400> 305

Be lie Tyr Pro Be Ser Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 306

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial clone 12CDHR2 synthetic HER2 ligand <400> 306

Be lie Tyr Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 307

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 13CDHR2 HER2 ligand <400> 307

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 308

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 14CDHR2 HER2 ligand <400> 308

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 309

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 15CDHR2 synthetic HER2 ligand <400> 309

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 310 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 16CDHR2 binder

<400> 310

Ser Tyr Pro Ser Tyr Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 311

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 17CDHR2 HER2 synthetic ligand

<400> 311

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 312

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder 18CDHR2 clone

<400> 312

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 313

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 19CDHR2 synthetic HER2 ligand

<400> 313

Be Tyr Tyr Pro Tyr Gly Be Tyr Tyr Wing Asp Ser Go Lys1 5 10 15Gly

<210> 314

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 20CDHR2 HER2 ligand <400> 314

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 315

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 21CDHR2 synthetic HER2 ligand <400> 315

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 316

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 22CDHR2 synthetic HER2 ligand <400> 316

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 317

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 23CDHR2 HER2 binder <400> 317Ser lie Tyr Pro Ser Tyr

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 318

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 318

24CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Tyr Tyr

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys

10 15

Gly

<210> 319

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 319

25CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Tyr Tyr

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 320

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 320

2 6CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Tyr Tyr1 5

Gly Be Thr Tyr Tyr Wing Asp Be Going Lys10 15

Gly

<210> 321

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial clone 27CDHR2 synthetic HER2 ligand <400> 321

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 322

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 28CDHR2 synthetic HER2 ligand <400> 322

Be lie Tyr Pro Be Tyr Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 323

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 29CDHR2 synthetic HER2 ligand <400> 323

Tyr lie Tyr Pro Be Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 324

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 30CDHR2 synthetic HER2 ligand <400> 324

Be lie Be Pro Tyr Tyr Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 325 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 31CDHR2 HER2 ligand <400> 325

Be lie Be Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 326

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 32CDHR2 synthetic HER2 ligand <400> 326

Be lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 327

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 33CDHR2 HER2 ligand <400> 327

Tyr lie Be Pro Be Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 328

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 34CDHR2 synthetic HER2 ligand <400> 328

Be lie Tyr Pro Be Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15Gly

<210> <211> <212> <213> 329 17 Artificial PRT

<220> <223> Artificial clone

<400> 329

Ser lie Tyr Pro Ser Ser 1 5 Gly

<210> <211> <212> <213> 330 17 Artificial PRT

<220> <223> Artificial clone

<400> 330

Ser lie Ser Pro Ser Ser 1 5 Gly

<210> <211> <212> <213> 331 17 Artificial PRT

<220> <223> Artificial clone

<400> 331

Ser lie Tyr Pro Ser Ser 1 5 Gly

<210> <211> <212> <213> 332 17 Artificial PRT

<220> <223> Artificial clone

35CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

36CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

37CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Tyr Tyr Wing Asp Will Go Lys10 15

38CDHR2 HER2 Synthetic Binder

<400>

332Be lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 333

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 39CDHR2 synthetic HER2 ligand <400> 333

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 334

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 40CDHR2 synthetic HER2 ligand <400> 334

Tyr lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 335

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 41CDHR2 synthetic HER2 ligand <400> 335

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 336

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 42CDHR2 HER2 ligand <400> 336

Tyr lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 337

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 43CDHR2 synthetic HER2 ligand <400> 337

Tyr lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 338

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 44CDHR2 synthetic HER2 ligand <400> 338

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 339

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 45CDHR2 HER2 ligand <400> 339

Ser lie Tyr Pro Ser Ser Gly Ser Thr Ser Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly <210> 340

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 46CDHR2 <400> 340

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 341

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 47CDHR2 ligand

<400> 341

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 342

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 binder synthetic clone 48CDHR2

<400> 342

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 343

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 49CDHR2

<400> 343Be lie Tyr Pro Ser Ser

1 5

Gly

<210> 344

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 344

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

50CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Ser Ser

Gly Tyr Thr Tyr Tyr Wing Asp Will Go Lys10 15

Gly

<210> 345

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 345

51CDHR2 HER2 Synthetic Binder

Tyr lie Tyr Pro Ser Ser

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 346

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 346

52CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Ser Tyr1 5

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 347

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 53CDHR2 HER2 ligand <400> 347

Tyr lie Tyr Pro Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 348

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 54CDHR2 synthetic HER2 ligand <400> 348

Be lie Tyr Pro Be Ser Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 349

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 55CDHR2 synthetic HER2 ligand <400> 349

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 350

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 56CDHR2 HER2 ligand <400> 350

Ser lie Tyr Pro Ser Ser Gly Ser Thr Ser Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 351 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 57CDHR2 HER2 ligand <400> 351

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 352

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 58CDHR2 ligand

<400> 352

Tyr lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 353

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 clone synthetic clone 59CDHR2

<400> 353

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 354

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 60CDHR2 binder

<400> 354

Tyr lie Ser Pro Ser Ser Gly Tyr Thr Ser Ser Tyr Ala Asp Ser Ser Lys1 5 10 15Gly

<210> 355

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 61CDHR2 HER2 ligand <400> 355

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 356

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 62CDHR2 synthetic HER2 ligand <400> 356

Tyr lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 357

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 63CDHR2 HER2 ligand <400> 357

Tyr lie Tyr Pro Be Gly Tyr Thr Tyr Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 358

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 64CDHR2 HER2 synthetic ligand <400> 358Ser lie Tyr Pro Ser Ser

1 5

Gly

<210> 359

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone

<400> 359

Ser lie: Ser Pro Ser Ser

Gly Be Thr Be Tyr Wing Asp Be Going Lys10 15

65CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 360

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 360

66CDHR2 HER2 Synthetic Binder

Ser lie Tyr Pro Ser Ser

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 361

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 361

67CDHR2 HER2 Synthetic Binder

Tyr lie Ser Pro Ser Ser 5

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

Gly

<210> 362

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial clone 68CDHR2 synthetic HER2 ligand <400> 362

Be lie Be Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 363

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 69CDHR2 synthetic HER2 ligand <400> 363

Tyr lie Tyr Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 364

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 70CDHR2 synthetic HER2 ligand <400> 364

Be lie Tyr Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 365

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 71CDHR2 synthetic HER2 ligand <400> 365

Be lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 366 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Artificial clone 72CDHR2 synthetic HER2 ligand <400> 366

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 367

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 73CDHR2 synthetic HER2 ligand

<400> 367

Be lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 368

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 74CDHR2

<400> 368

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 369

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 75CDHR2 HER2 ligand

<400> 369

Be Tyr Tyr Pro Tyr Gly Be Thr Be Tyr Ala Asp Be Go Lys1 5 10 15Gly

<210> 370

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 76CDHR2 synthetic HER2 ligand <400> 370

Tyr lie Tyr Pro Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 371

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 77CDHR2 synthetic HER2 ligand <400> 371

Tyr lie Be Pro Be Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 372

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 78CDHR2 synthetic HER2 ligand <400> 372

Be lie Be Pro Be Be Gly Be Thr Be Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 373

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 79CDHR2 synthetic HER2 ligand <400> 373Ser lie Ser Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys1 5 10 15

Gly

<210> 374

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 80CDHR2 synthetic HER2 ligand <400> 374

Be lie Tyr Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 375

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 81CDHR2 synthetic HER2 ligand <400> 375

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 376

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 82CDHR2 HER2 ligand <400> 376

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 377

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 83CDHR2 HER2 ligand <400> 377

Be Ile Be Pro Be Ser Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 378

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 84CDHR2 synthetic HER2 ligand <400> 378

Tyr Ile Be Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 379

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 85CDHR2 synthetic HER2 ligand <400> 379

Tyr Ile Be Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 380

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 86CDHR2 synthetic HER2 ligand <400> 380

Tyr Ile Tyr Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 381 <211> 17 <212> <213> Artificial PRT

<220> <223> Artificial clone

<400> 381

Ser lie Ser Pro Ser Tyr 1 5 Gly

<210> <211> <212> <213> 382 17 Artificial PRT

<220> <223> Artificial clone

<400> 382

Ser lie Ser Pro Ser Tyr 1 5 Gly

<210> <211> <212> <213> 383 17 Artificial PRT

<220> <223> Artificial clone

<400> 383

Ser lie Tyr Pro Tyr Ser 1 5 Gly

<210> <211> <212> <213> 384 17 Artificial PRT

<220> <223> Artificial clone

<400> 384

87CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

88CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

8 9CDHR2 HER2 Synthetic Binder

Gly Tyr Thr Be Tyr Wing Asp Be Going Lys10 15

90CDHR2 HER2 Synthetic Binder

Be lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys15 10 15Gly

<210> 385

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 91CDHR2 synthetic HER2 ligand <400> 385

Tyr lie Tyr Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 386

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 ligand clone 92CDHR2 <400> 386

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 387

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 93CDHR2 synthetic HER2 ligand <400> 387

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 388

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 94CDHR2 synthetic HER2 ligand <400> 388Tyr lie Ser Pro Ser Tyr Gly Ser Thr Tyr Ala Asp Ser Val Lys1 5 10 15

Gly

<210> 389

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 95CDHR2 synthetic HER2 ligand <400> 389

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 390

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder 96CDHR2 clone <400> 390

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 391

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 97CDHR2 synthetic HER2 ligand <400> 391

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 392

<211> 17

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic HER2 binder 98CDHR2 clone <400> 392

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 393

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 99CDHR2 synthetic HER2 ligand <400> 393

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 394

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 100CDHR2 synthetic HER2 ligand <400> 394

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 395

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 101CDHR2 synthetic HER2 ligand <400> 395

Ser Tyr Pro Ser Tyr Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 396 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 ligand clone 102CDHR2 <400> 396

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 397

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 103CDHR2 HER2 ligand <400> 397

Ser Tyr Tyr Pro Tyr Gly Ser Tyr Tyr Ala Asp Ser Go Lys1 5 10 15

Gly

<210> 398

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 104CDHR2 synthetic HER2 ligand <400> 398

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 399

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 105CDHR2 synthetic HER2 ligand <400> 399

Be lie Tyr Pro Be Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15Gly

<210> 400

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 binder synthetic clone 106CDHR2

<400> 400

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 401

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 binder synthetic clone 106CDHR2

<400> 401

Tyr Tyr Ser Ser Gly Phe Asp Tyr1 5

<210> 402

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 1CDHR3 HER2 synthetic binder

<400> 402

Tyr Tyr Gly Tyr Gly Met Asp Tyr1 5

<210> 403

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 3CDHR3 ligand

<400> 403

Tyr Tyr Gly Tyr Gly Read Asp Tyr1 5 <210> 404

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 4CDHR3 HER2 synthetic ligand

<400> 404

Tyr Tyr Gly Tyr Gly Phe Asp Tyr

<210> 405

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 5CDHR3 ligand

<400> 405

Tyr Tyr Gly Ser Gly Phe Asp Tyr

<210> 406

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 6CDHR3 ligand

<400> 406

Tyr Tyr Gly Gly Wing Phe Asp Tyr

<210> 407

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial HER2 synthetic clone 7CDHR3 ligand

<400> 407

Tyr Tyr Gly Arg Gly Wing Met Asp Tyr1 5

<210> 408

<211> 9

<212> PRT

<213> Artificial <220>

<223> Artificial clone 8CDHR3 synthetic HER2 ligand <400> 408

Tyr Ser Tyr Ser Gly Wing Read Asp Tyr1 5

<210> 409

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 9CDHR3 synthetic HER2 ligand <400> 409

Tyr Arg Tyr Tyr Gly Wing Read Asp Tyr1 5

<210> 410

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 10CDHR3 synthetic HER2 ligand <400> 410

Tyr Arg Tyr Gly Gly Wing Met Asp Tyr1 5

<210> 411

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 11CDHR3 HER2 ligand <400> 411

Tyr Tyr Gly Gly Tyr Gyr Tyr Gly Met Asp Tyr1 5 10

<210> 412

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 12CDHR3 synthetic HER2 ligand <400> 412

Tyr Ser Tyr Tyr Tyr Tyr Wing Read Asp Tyr1 5 10

<210> 413

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 13CDHR3 HER2 ligand <400> 413

Being Being Tyr Tyr Tyr Tyr Gly Met Asp Tyr1 5 10

<210> 414

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 14CDHR3 HER2 ligand <400> 414

Being Being Tyr Tyr Tyr Tyr Gly Phe Asp Tyr1 5 10

<210> 415

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 15CDHR3 synthetic HER2 ligand <400> 415

Ser Ser Tyr Tyr Tyr Tyr Gly Read Asp Tyr1 5 10

<210> 416

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 16CDHR3 HER2 synthetic ligand <400> 416

Ser Gly Gly Tyr Ser Arg Gly lie Asp Tyr1 5 10 <210> 417

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 17CDHR3 synthetic HER2 ligand <400> 417

Gly Tyr Ser Tyr Ser Arg Gly lie Asp Tyr1 5 10

<210> 418

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 18CDHR3 synthetic HER2 ligand <400> 418

Gly Tyr Be Tyr Be Arg Gly Met Asp Tyr1 5 10

<210> 419

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 19CDHR3 synthetic HER2 ligand <400> 419

Gly Ser Tyr Ser Tyr Gly Met Asp Tyr1 5 10

<210> 420

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 20CDHR3 HER2 ligand <400> 420

Gly Ser Gly Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 421

<211> 10

<212> PRT

<213> Artificial <220>

<223> Artificial synthetic clone 21CDHR3 HER2 ligand <400> 421

Gly Ser Gly Tyr Gly Tyr Gly lie Asp Tyr1 5 10

<210> 422

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 22CDHR3 <400> 422

Gly Arg Arg Tyr Ser Arg Gly lie Asp Tyr1 5 10

<210> 423

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 23CDHR3 HER2 ligand <400> 423

Gly Gly Being Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 424

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 24CDHR3 HER2 ligand <400> 424

Gly Gly Being Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 425

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 25CDHR3 synthetic HER2 ligand <400> 425Gly Gly Arg Tyr Gly Tyr Gly Leu Asp Tyr1 5 10

<210> 426

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 26CDHR3 synthetic HER2 ligand <400> 426

Gly Gly Arg Tyr Gly Gly Arg Gly lie Asp Tyr1 5 10

<210> 427

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 27CDHR3 synthetic HER2 ligand <400> 427

Gly Gly Tly Gly Tyr Gly Tyr Gly lie Asp Tyr1 5 10

<210> 428

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 28CDHR3 HER2 ligand <400> 428

Gly Gly Tly Gly Tyr Gly Tyr Gly lie Asp Tyr1 5 10

<210> 429

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 29CDHR3 synthetic HER2 ligand <400> 429

Tyr Tyr Tyr Being Gly Gly Being Tyr Gly Met Asp Tyr1 5 10

<210> 430 <211> 12

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 30CDHR3 synthetic HER2 ligand <400> 430

Being Tyr Being Being Tyr Tyr Being Gly Met Asp Tyr1 5 10

<210> 431

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 31CDHR3 synthetic HER2 ligand <400> 431

Arg Tyr Tyr Tyr Tyr Tyr Ser Ala Phe Asp Tyr1 5 10

<210> 432

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 32CDHR3 synthetic HER2 ligand <400> 432

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Gly Ala lie Asp Tyr1 5 10

<210> 433

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 33CDHR3 HER2 ligand <400> 433

Ser Tyr Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Gly Met Asp Tyr1 5 10

<210> 434

<211> 14

<212> PRT

<213> Artificial

<220> <223> Artificial clone 34CDHR3 synthetic HER2 ligand <400> 434

Be Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Gly Read Asp Tyr1 5 10

<210> 435

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 35CDHR3 HER2 ligand <400> 435

Ser Tyr Tyr Tyr Ser Tyr Tyr Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 436

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 36CDHR3 synthetic HER2 ligand <400> 436

Be Tyr Tyr Gly Be Tyr Tyr Gly Tyr Gly Phe Asp Tyr1 5 10

<210> 437

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 37CDHR3 synthetic HER2 ligand <400> 437

Ser Tyr Tyr Gly Tyr Tyr Ser Gly Tyr Gly lie Asp Tyr1 5 10

<210> 438

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 38CDHR3 synthetic HER2 ligand <400> 438Ser Ser Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Gly Leu Asp Tyr1 5 10

<210> 439

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 39CDHR3 synthetic HER2 ligand <400> 439

Gly Tyr Tyr Tyr Ser Tyr Tyr Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 440

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 40CDHR3 HER2 synthetic ligand <400> 440

Gly Tyr Tyr Tyr Be Tyr Tyr Tyr Gly Ser Ala Met Asp Tyr1 5 10

<210> 441

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 41CDHR3 synthetic HER2 ligand <400> 441

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Gly Met Asp Tyr1 5 10

<210> 442

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 42CDHR3 synthetic HER2 ligand <400> 442

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Wing Read Asp Tyr1 5 10

<210> 443 <211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 43CDHR3 HER2 ligand <400> 443

Gly Tyr Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Ala lie Asp Tyr1 5 10

<210> 444

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 44CDHR3 HER2 ligand <400> 444

Gly Tyr Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Ala Met Asp Tyr1 5 10

<210> 445

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 45CDHR3 synthetic HER2 ligand <400> 445

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Gly lie Asp Tyr1 5 10

<210> 446

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 46CDHR3 HER2 ligand <400> 446

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Gly Read Asp Tyr1 5 10

<210> 447

<211> 14

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic HER2 ligand clone 47CDHR3

<400> 447

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Tyr Gly Met Asp Tyr1 5 10

<210> 448

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 48CDHR3 HER2 ligand <400> 448

Gly Tyr Tyr Tyr Be Tyr Tyr Be Gly Ser Gly Read Asp Tyr1 5 10

<210> 449

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 49CDHR3 HER2 ligand <400> 449

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 450

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 50CDHR3 HER2 ligand <400> 450

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly lie Asp Tyr1 5 10

<210> 451

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 51CDHR3 HER2 synthetic ligand <400> 451Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 452

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 52CDHR3 HER2 ligand <400> 452

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 453

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 53CDHR3 HER2 ligand <400> 453

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 454

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 54CDHR3 synthetic HER2 ligand <400> 454

Gly Tyr Tyr Tyr Be Tyr Tyr Gly Gly Tyr Gly Read Asp Tyr1 5 10

<210> 455

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 55CDHR3 synthetic HER2 ligand <400> 455

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 456 <211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 56CDHR3 HER2 ligand <400> 456

Gly Tyr Tyr Tyr Ser Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 457

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 57CDHR3 synthetic HER2 ligand <400> 457

Gly Tyr Tyr Tyr Gly Tyr Tyr Ser Gly Tyr Gly lie Asp Tyr1 5 10

<210> 458

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 58CDHR3 synthetic HER2 ligand <400> 458

Gly Tyr Tyr Tyr Tyr Tyr Gly Tyr Gly Met Asp Tyr1 5 10

<210> 459

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 59CDHR3 synthetic HER2 ligand <400> 459

Gly Ser Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Ala lie Asp Tyr1 5 10

<210> 460

<211> 14

<212> PRT

<213> Artificial

<220> <223> Artificial clone 60CDHR3 synthetic HER2 ligand <400> 460

Gly Ser Tyr Tyr Ser Tyr Tyr Ser Gly Tyr Ala Phe Asp Tyr1 5 10

<210> 461

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 61CDHR3 <400> 461

Gly Ser Tyr Tyr Ser Tyr Tyr Ser Gly Ser Tyr1 Asp Tyr1 5 10

<210> 462

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 62CDHR3 <400> 462

Gly Ser Tyr Tyr Gly Tyr Tyr Tyr Gly Ser Ala lie Asp Tyr1 5 10

<210> 463

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 63CDHR3 HER2 ligand <400> 463

Gly Ser Tyr Tyr Gly Ser Tyr Tyr Gly Ser Gyr Met Asp Tyr1 5 10

<210> 464

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 64CDHR3 HER2 synthetic ligand <400> 464Gly Ser Tyr Tyr Gly Tyr Ser Gly Ser Gly Met Asp Tyr1 5 10

<210> 465

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 65CDHR3 HER2 synthetic ligand <400> 465

Gly Be Gly Tyr Be Tyr Tyr Gly Gly Tyr Gly Read Asp Tyr1 5 10

<210> 466

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 66CDHR3 HER2 ligand <400> 466

Gly Tyr Tyr Gly Tyr Tyr Tyr Gly Tyr Gly Read Asp Tyr1 5 10

<210> 467

<211> 14

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 67CDHR3 HER2 ligand <400> 467

Gly Arg Tyr Being Gly Tyr Tyr Gly Gly Tyr Gly Met Asp Tyr1 5 10

<210> 468

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 68CDHR3 synthetic HER2 ligand <400> 468

Tyr Tyr Tyr Ser Ser Gly Tyr Tyr Tyr Tyr Tyr Ala Phe Asp Tyr1 5 10 15

<210> 469 <211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 69CDHR3 synthetic HER2 ligand <400> 469

Tyr Be Tyr Be Tyr Tyr Gly Tyr Tyr Gly Be Gly Met Asp Tyr1 5 10 15 <210> 470

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 70CDHR3 HER2 synthetic ligand <400> 470

Tyr Arg Be Tyr Tyr Be Tyr Arg Tyr Gly Tyr Gly Met Asp Tyr1 5 10 15

<210> 471

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 71CDHR3 synthetic HER2 ligand <400> 471

Tyr Gly Tyr Tyr Tyr Be Tyr Tyr Gly Gly Be Gly Read Asp Tyr1 5 10 15

<210> 472

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 72CDHR3 synthetic HER2 ligand <400> 472

Tyr Gly Tyr Tyr Tyr Be Tyr Tyr Gly Gly Be Gly Read Asp Tyr1 5 10 15

<210> 473

<211> 15

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 73CDHR3 HER2 ligand <400> 473

Tyr Gly Tyr Ser Tyr Ser Tyr Ser Gyr Ser Ala Leu Asp Tyr1 5 10 15

<210> 474

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 74CDHR3 HER2 synthetic ligand <400> 474

Be Tyr Tyr Tyr Gly Gly Tyr Tyr Be Gly Tyr Gly Met Asp Tyr1 5 10 15

<210> 475

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 75CDHR3 HER2 synthetic ligand <400> 475

Arg Arg Be Tyr Tyr Be Tyr Arg Tyr Be Tyr Gly Read Asp Tyr1 5 10 15

<210> 476

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 76CDHR3 HER2 synthetic ligand <400> 476

Arg Arg Be Tyr Tyr Be Tyr Be Arg Be Tyr Ala Met Asp Tyr1 5 10 15

<210> 477

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 77CDHR3 HER2 synthetic ligand <400> 477Tyr Tyr Tyr Gyr Tyr Tyr Tyr Ser Tyr Tyr Gly Leu Asp Tyr1 5 10 15

<210> 478

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 78CDHR3 synthetic HER2 ligand <400> 478

Tyr Tyr Gyr Tyr Gyr Tyr Gyr Tyr Gyr Gly Gly Gyr Read Asp Tyr1 5 10 15

<210> 479

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 79CDHR3 synthetic HER2 ligand <400> 479

Tyr Tyr Tyr Gly Tyr Tyr Ser Tyr Tyr Gly Gly Ser Ala Met Asp Tyr1 5 10 15

<210> 480

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 80CDHR3 synthetic HER2 ligand <400> 480

Tyr Tyr Tyr Tyr Gly Gly Tyr Tyr Tyr Tyr Tyr Tyr Ala Phe Asp Tyr1 5 10 15

<210> 481

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 81CDHR3 synthetic HER2 ligand <400> 481

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Gly lie Asp Tyr1 5 10 15

<210> 482 <211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 82CDHR3 HER2 ligand <400> 482

Tyr Tyr Be Tyr Tyr Be Tyr Tyr Tyr Tyr Tyr Gly Gly Met Asp Tyr1 5 10 15

<210> 483

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 83CDHR3 HER2 ligand <400> 483

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Ala Met Asp Tyr1 5 10 15

<210> 484

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic clone 84CDHR3 HER2 ligand <400> 484

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Tyr Gly Phe Asp Tyr1 5 10 15

<210> 485

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder clone 85CDHR3 <400> 485

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Tyr Gly Phe Asp Tyr1 5 10 15

<210> 486

<211> 16

<212> PRT

<213> Artificial

<220> <223> Artificial clone 86CDHR3 synthetic HER2 ligand <400> 486

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Gly Ala Phe Asp Tyr1 5 10 15

<210> 487

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 87CDHR3 synthetic HER2 ligand <400> 487

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Gly Gly Met Asp Tyr1 5 10 15

<210> 488

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 88CDHR3 HER2 synthetic ligand <400> 488

Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Gly Gly Read Asp Tyr1 5 10 15

<210> 489

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 89CDHR3 synthetic HER2 ligand <400> 489

Tyr Tyr Tyr Tyr Tyr Gly Tyr Tyr Gyr Tyr Gly Read Asp Tyr1 5 10 15

<210> 490

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 90CDHR3 synthetic HER2 ligand <400> 490Tyr Ser Ser Tyr Tyr Tyr

Ser Tyr Tyr Ser Gly Ser Gly Read Asp Tyr10 15

<210> 491

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 491

91CDHR3 HER2 Synthetic Binder

Ser Ser Tyr Tyr Tyr Tyr

Ser Tyr Tyr Gly Gly Ser Gly lie Asp Tyr10 15

<210> 4 92

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 492

92CDHR3 HER2 Synthetic Binder

Arg Ser Tyr Tyr Tyr Tyr

Ser Tyr Tyr Tyr Ser Arg Wing Met Asp Tyr10 15

<210> 493

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 493

93CDHR3 HER2 Synthetic Binder

Ser Tyr Ser Ser Tyr Tyr

Being Tyr Tyr Being Tyr Gly Gly Met Asp10 15

Tyr

<210> 494

<211> 21

<212> PRT

<213> Artificial

<220>

<223> Artificial

<400> 494

94CDHR3 HER2 Synthetic Binder

Tyr Arg Tyr Tyr Tyr Be Arg Tyr Gly Tyr Arg Tyr Tyr Tyr Tyr Tyr15 10 15Arg Wing Read Asp Tyr20

<210> 495 <211> 8 <212> PRT <213> Artificial

<220>

<223> Artificial clone 95CDHR3 synthetic HER2 ligand <400> 495

Arg Tyr Be Ser Gly Met Asp Tyr1 5

<210> 496

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 96CDHR3 synthetic HER2 ligand <400> 496

Tyr Be His Ser Gly Wing Phe Asp Tyr1 5

<210> 497

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 97CDHR3 synthetic HER2 ligand <400> 497

Gly Tyr Ser Tyr Gly His Gly lie Asp Tyr15 10

<210> 498

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial synthetic HER2 binder 98CDHR3 clone <400> 498

Gly Being Ser Phe Gly Arg Gly Phe Asp Tyr15 10

<210> 499 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 99CDHR3 synthetic HER2 ligand <400> 499

Gly Be Ser Tyr Be Trp Gly Read Asp Tyr15 10

<210> 500

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 100CDHR3 synthetic HER2 ligand <400> 500

Gly Be Arg Tyr Be His Gly Met Asp Tyr15 10

<210> 501

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 101CDHR3 synthetic HER2 ligand <400> 501

Gly Wing Ser Tyr Gly Tyr Gly Met Asp Tyr15 10

<210> 502

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 102CDHR3 synthetic HER2 ligand <400> 502

Gly Met Being Tyr Gly Tyr Gly Met Asp Tyr15 10

<210> 503

<211> 10

<212> PRT

<213> Artificial

<220> <223> Artificial synthetic clone 103CDHR3 HER2 ligand <400> 503

Gly Gly Arg Tyr Asn His Gly Read Asp Tyr15 10

<210> 504

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 104CDHR3 synthetic HER2 ligand <400> 504

Glu Tyr Tyr Gln Gly Tyr Gly Pro Tyr Arg Ser Thr Tyr Gly Leu Asp15 10 15

Tyr

<210> 505

<211> 19

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 105CDHR3 synthetic HER2 ligand <400> 505

Be Be Trp Be Be Arg Gly Be Be Tyr Be Arg Thr Wing Gly Gly15 10 15

Met Asp Tyr

<210> 506

<211> 20

<212> PRT

<213> Artificial

<220>

<223> Artificial clone 106CDHR3 synthetic HER2 ligand <400> 506

Glu Gly Tyr Tyr Be Will Be Gly Be Tyr Be Tyr Be Thr Arg Gly15 10 15

Gly Pro Asp Tyr20

<210> 507 <211> 9 <212> PRT <213> Artificial <220>

<223> Synthetic DR5 Clone 1CDRL3 Binder

<400> 507

Gln Gln Tyr Be Ser Tyr Pro Ser Thr1 5

<210> 508

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 2CDRL3 Clone

<400> 508

Gln Gln Tyr Be Ser Tyr Pro Ser Thr1 5

<210> 509

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 3CDRL3

<400> 509

Gln Tyr Tyr Tyr Tyr Tyr Pro Ser Thr1 5

<210> 510

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Clone 4CDRL3 Binder

<400> 510

Gln Gln Tyr Be Ser Tyr Pro Ser Thr1 5

<210> 511

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 5CDRL3 clone <400> 511

Gln Gln His Tyr Thr Thr Pro Pro Thr1 5

<210> 512

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Clone 6CDRL3 Binder

<400> 512

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 513

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Clone 7CDRL3 Binder

<400> 513

Gln Gln Tyr Ser Tyr Ser Pro 5

<210> 514

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 8CDRL3 Clone

<400> 514

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 515

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 9CDRL3

<400> 515

Gln Gln Tyr Ser Ser Ser Ser Pro Thr1 5 <210> 516

<211> 9

<212> PRT

<213> Artificial

<220>

<223> DR5 Synthetic DR5 Binder Clone 10CDRL3

<400> 516

Gln Gln His Tyr Thr Thr Pro Pro Thr1 5

<210> 517

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 11CDRL3 Clone

<400> 517

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 518

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Clone 12CDRL3 Binder

<400> 518

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 519

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Clone 13CDRL3 Binder

<400> 519

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 520

<211> 9

<212> PRT

<213> Artificial <220>

<223> Synthetic DR5 Binder 14CDRL3 Clone

<400> 520

Gln Gln Tyr Ser Ser Ser Ser Pro 5

<210> 521

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 15CDRL3

<400> 521

Gln Gln Be Ser Tyr Ser Pro Ser Thr1 5

<210> 522

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 16CDRL3 Clone

<400> 522

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 523

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 17CDRL3 Clone

<400> 523

Gln Gln Tyr Tyr Ser Ser Ser Pro 5

<210> 524

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 1CDRH1

<400> 524Gly Phe Ser lie Tyr Ser Tyr

1 5

<210> <211> <212> <213> 525 10 Artificial PRT

<220> <223> Binding 2CDRH1 Clone

<400> 525

Gly Phe Ser lie Tyr 1 5 Ser Ser

<210> <211> <212> <213> 526 10 Artificial PRT

<220> <223> Binding clone 3CDRH1

<400> 526

Gly Phe Tyr lie Tyr 1 5 Ser Tyr

<210> <211> <212> <213> 527 10 Artificial PRT

<220> <223> 4CDRH1 Binding Clone

<400> 527

Ser lie His10

synthetic DR5

Ser lie His10

synthetic DR5

Ser lie His10

synthetic DR5

Gly Phe Ser

Ser lie His10

<210> 528

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Clone 5CDRH1

<400> 528

synthetic DR5 binder

Gly Phe Ser Tyr Ser 5

Ser lie His10

<210> 529 <211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 6CDRH1 clone <400> 529

Gly Phe Tyr lie Ser Tyr Ser Ser His15 10

<210> 530

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 7CDRH1 clone <400> 530

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 531

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 8CDRH1 Clone <400> 531

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 10

<210> 532

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 9CDRH1 clone <400> 532

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 533

<211> 10

<212> PRT

<213> Artificial

<220> <223> Synthetic DR5 binder 10CDRH1 clone <400> 533

Gly Phe Tyr lie Tyr Ser Ser Ser Ser His15 10

<210> 534

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 11CDRH1 clone <400> 534

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 535

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Clone 12CDRH1 synthetic DR5 linker <400> 535

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 536

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 13CDRH1 clone <400> 536

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 537

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 14CDRH1 Clone

<400> 537Gly Phe Tyr Ile Being Ser Being Tyr Ile His15 10

<210> 538

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 15CDRH1 clone <400> 538

Gly Phe Tyr Ile Tyr Ser Ser Ser Ser Ile His15 10

<210> 539

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 16CDRH1 Clone <400> 539

Gly Phe Tyr Ile Tyr Ser Ser Ser Ser Ile His15 10

<210> 540

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Bind 17CDRH1 Clone <400> 540

Gly Phe Tyr Ile Being Being Being Being Ile His15 10

<210> 541

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 18CDRH1 Clone <400> 541

Be Ile Be Pro Tyr Tyr Gly Tyr Thr Be Tyr Wing Asp Be Go Lys15 10 15

Gly <210> 542

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 2CDRH2 clone <400> 542

Be lie Tyr Pro Tyr Be Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 543

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 3CDRH2 <400> 543

Be lie Be Pro Tyr Tyr Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 544

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 4CDRH2 clone <400> 544

Tyr lie Be Pro Be Tyr Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 545

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 5CDRH2 clone <400> 545

Be lie Be Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15Gly

<210> 546

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 6CDRH2 clone <400> 546

Tyr lie Be Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 547

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 7CDRH2 clone <400> 547

Be lie Be Pro Be Tyr Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 548

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 8CDRH2 Clone <400> 548

Be lie Be Pro Be Be Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 549

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 9CDRH2 <400> 549See lie Pro Tyr Tyr Gly Tyr Thr Tyr Ala Asp Ser Vai Lys1 5 10 15

Gly

<210> 550

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 10CDRH2 clone <400> 550

Be lie Be Pro Be Be Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 551

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 11CDRH2 clone <400> 551

Tyr lie Be Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 552

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 12CDRH2 clone <400> 552

Tyr lie Tyr Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 553

<211> 17

<212> PRT

<213> Artificial

<220> <223> Synthetic DR5 Binder 13CDRH2 Clone <400> 553

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 554

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 14CDRH2 clone <400> 554

Be lie Be Pro Be Tyr Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 555

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 15CDRH2 clone <400> 555

Be lie Be Pro Be Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 556

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 16CDRH2 Clone <400> 556

Tyr lie Be Pro Tyr Tyr Gly Be Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 557 <211> 17 <212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 17CDRH2 Clone <400> 557

Tyr lie Be Pro Be Tyr Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 558

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 1CDRH3 Clone <400> 558

Arg Tyr Tyr Ser Tyr Arg Ser Tyr Ser Tyr Ser Tyr Ser Gly Leu Asp Tyr1 5 10 15

<210> 559

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 2CDRH3 clone <400> 559

Arg Tyr Tyr Be Ser Be Tyr Arg Be Ser Be Tyr Gly Met Asp Tyr1 5 10 15

<210> 560

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder Clone 3CDRH3 <400> 560

Tyr Tyr Tyr Be Tyr Tyr Tyr Tyr Gly Be Tyr Tyr Gly Read Asp Tyr1 5 10 15

<210> 561

<211> 18

<212> PRT

<213> Artificial <220>

<223> Synthetic DR5 binder 4CDRH3 clone <400> 561

Arg Arg Tyr Be Arg Tyr Tyr Arg Tyr Be Ser Arg Tyr Arg Gly Phe1 5 10 15

Asp Tyr

<210> 562

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 5CDRH3 clone <400> 562

Tyr Arg Be Tyr Arg Tyr Gly Tyr Tyr Arg Gly Gly Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 563

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 6CDRH3 clone <400> 563

Tyr Arg Be Tyr Arg Tyr Gly Tyr Tyr Arg Gly Be Tyr Wing Read Asp1 5 10 15

Tyr

<210> 564

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 7CDRH3 clone <400> 564

Tyr Arg Be Tyr Arg Tyr Gly Be Tyr Arg Gly Be Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 565 <211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 8CDRH3 Clone <400> 565

Tyr Arg Be Tyr Arg Tyr Gly Be Tyr Tyr Gly Be Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 566

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 9CDRH3 clone <400> 566

Tyr Arg Tyr Arg Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 567

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 10CDRH3 clone <400> 567

Tyr Arg Tyr Arg Tyr Tyr Gly Tyr Tyr Be Gly Gly Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 568

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 11CDRH3 clone <400> 568

Tyr Arg Arg Tyr Arg Tyr Gly Be Tyr Tyr Be Gly Tyr Ala Phe Asp1 5 10 15Tyr

<210> 569

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 12CDRH3 Clone <400> 569

Tyr Arg Tyr Arg Tyr Tyr Gly Tyr Tyr Gly Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 570

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Clone 13CDRH3 synthetic DR5 linker <400> 570

Gyr Tyr Arg Gyr Tyr Gyr Tyr Gyr Tyr Gyr Gyr Tyr Ala Phe Asp1 5 10 15

Tyr

<210> 571

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 binder 14CDRH3 clone <400> 571

Arg Arg Tyr Tyr Arg Tyr Gly Tyr Be Arg Gly Be Tyr Gly Read Asp1 5 10 15

Tyr

<210> 572

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Clone 15CDRH3 synthetic DR5 binder <400> 572Arg Arg Ser Tyr Arg Tyr Gly Ser Tyr Arg Gly Ser Tyr Ala Phe Asp

1 5 10 15

Tyr

<210> 573

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 16CDRH3 Clone

<400> 573

Arg Arg Pro Tyr Arg Tyr Gly Arg Be Arg Gly Tyr Tyr Ala Phe Asp

1 5 10 15

Tyr

<210> 574

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Synthetic DR5 Binder 17CDRH3 Binder

<400> 574

Met Arg Arg Tyr Gyr Tyr Gly Arg Tyr Ser Gly Ala Tyr Gly Leu Asp

1 5 10 15

Tyr

<210> 575

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is R or Y

<220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is Y or R

<220>

<221> MISC_FEATURE

<222> (14). . (14) <223> X is R or Y <400> 575

Xaa Arg Be Tyr Arg Tyr Gly Be Tyr Xaa Gly Be Tyr Xaa Phe Asp1 5 10 15

Tyr

<210> 576

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 576

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 577

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 577

Be lie Be Pro Be Be Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 578

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 578

Tyr Arg Be Tyr Arg Tyr Gly Be Tyr Tyr Gly Be Tyr Gly Phe Asp1 5 10 15

Tyr

<210> 579

<211> 10

<212> PRT

<213> Artificial <220>

<223> synthetic CDRH1

<400> 579

Gly Phe Tyr Ile Tyr Ser Ser Ser Ser Ile His1 5 10

<210> 580

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 580

Be Ile Be Pro Be Ser Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 581

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 581

Arg Arg Be Tyr Arg Tyr Gly Be Tyr Arg Gly Be Tyr Ala Phe Asp15 10 15

Tyr

<210> 582

<211> 9

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is R or Y

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is R, S, or Y <220> <221> MISC_FEATURE

<222> (3) .. (3)

<223> X is S, G, Y, or H

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is S, G, Y, or R

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is F, M, L, or A

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> X is F, M, L,

<400> 582

Xaa Xaa Xaa Xaa Xaa1 5

<210> 583

<211> 10

<212> PRT

<213> Artificial

or no amino acid

Xaa Xaa Asp Tyr

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is G, S, or Y

<220>

<221> MI SC_FEATURE

<222> (2) .. (2)

<223> X is Y, S, G, R, A, or M

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is G, R, S, or Y

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is F, G, or Y <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is G, N, S, or Y

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is H, R, W, or Y

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> X is A or G

<220>

<221> MISC_FEATURE

<222> (8). . (8)

<223> X is F, I, L, or M

<400> 583

Xaa_Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr

1 5 10

<210> 584

<211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y, S, R, G, or E

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is Y, S, R, or G

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is S, Y, G, or W

<220>

<221> MISC_FEATURE

<222> (4). . (4)

<223> X is S, Y, G, or W

<220><221> <222>

MISC_FEATURE (5). . (5) <223> X is G, Y, or S <220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is G, Y, S, R, or V

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> X is S, Y, G, or R

<220>

<221> MISC_FEATURE <222> (8) .. (8)

<223> X is Y, S, G, R, P, or V

<220>

<221> MISC_FEATURE

<222> (9) .. (9)

<223> X is G, A, R, S, or Y

<220>

<221> MISC_FEATURÊ

<222> (10) .. (10)

<223> X is M, F, G, Y, S, or R

<220>

<221> MISC_FEATURE

<222> (11) .. (11)

<223> X is A, G, R, S, Y, or no amino acids

<220>

<221> MISC_FEATURE

<222> (12). . (12)

<223> X is A, F, G, I, L, M, R, S, T, Y, or no amino acids

<220>

<221> MISC_FEATURE

<222> (13) .. (13)

<223> X is A, F, G, L, M, S, T, Y, or no amino acid <220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is L, M, F, I, G, Y, A, T, or no amino acid <220>

<221> MISC_FEATURE

<222> (15) .. (15)

<223> X is M, L, Y, G, R, or no amino acid <220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is G, Y, or no amino acid <220>

<221> MISC_FEATURE

<222> (17) .. (17)

<223> X is G, R, M, or no amino acid <220>

<221> MISC_FEATURE

<222> (18) .. (18)

<223> X is A, P, or no amino acid <220>

<221> MISC_FEATURE

<222> (19) .. (19)

<223> X is L or no amino acids

<400> 584

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa15 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 585

<211> 6

<212> DNA

<213> Artificial

<220>

<223> Synthetic transcription initiation upstream sequence <220>

<221> misc_feature

<222> (2) .. (2)

<223> N cannot be any nucleotide

<400> 585

cncaat 6

<210> 586

<211> 6

<212> DNA

<213> Artificial

<220>

<223> Synthetic Sign

<400> 586

aataaa 6

<210> 587

<211> 9

<212> PRT

<213> Artificial

<220> <223> Synthetic CDRL3 <220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is Y or S

<400> 587

Gln Gln Ser Tyr Tyr Xaa Pro Ser Thr

<210> 588

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is independently Y or S

<400> 588

Gly Phe Ser Ile Xaa Xaa Ser Tyr Ile His1 5 10 <210> 589

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S

<400> 589

Ser Ile Tyr Pro Xaa Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Lys Vai1 5 10 15

Gly

<210> 590 <211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is either Y or S

<400> 590

Gly Phe Xaa Ile Be Tyr Be Ser Ile His1 5 10

<210> 591

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (8). . (8)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is independently Y or S

<400> 591

Ser Ile Tyr Pro Xaa Tyr Gly Xaa Thr Xaa Tyr Wing Asp Ser Lys Vai15 10 15

Gly

<210> 592

<211> 16

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <220> <221> MISC_FEATURE

<222> (1). . (1)

<223> X is Y, S, or R

<220>

<221> MISC_FEATURE <222> (2) .. (2)

<223> X is Y or S

<220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is G, Y, or S

<220>

<221> MISC_FEATURE

<222> (4). . (4)

<223> X is Y, S, R, or G

<220>

<221> MISC_FEATURE

<222> (10). . (10)

<223> X is Y, S, or G

<220>

<221> MISC_FEATURE

<222> (12). . (12)

<223> X is Y, S, G, or R

<220>

<221> MISC_FEATURE

<222> (13). . (13)

<223> X is G or A

<220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is M, I, F, or L

<400> 592

Xaa Xaa Xaa Xaa Tyr Tyr Ser Tyr1 5

<210> 593

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (3). . (3)

<223> X is Y or S

Tyr Xaa Gly Xaa Xaa Xaa Asp Tyr10 15 <400> 593

Gly Phe Xaa Ile Being Being Being Being Ile His1 5 10

<210> 594

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (1). . (1)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (3). . (3)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is independently Y or S

<400> 594

Xaa Ile Xaa Pro Be Ser Gly Tyr Thr Xaa Tyr Ala Asp Ser Lys Vai15 10 15

Gly

<210> 595

<211> 184

<212> PRT

<213> homo

<400> 595

sapiens

Met Ser Ala Leu Leu Ile Leu Ala Leu Go Gly Wing Go Wing Asp15 10 15

Tyr Lys Asp Asp Asp Asp Lys Read Sera Wing Read Ile Thr Gln Gln Asp20 25 30

Read Wing Pro Gln Gln Arg Go Wing Pro Gln Gln Lys Arg Ser Ser Pro35 40-45

Be Glu Gly Leu Cys Pro Gly His His Ile Be Glu Asp Gly Arg50 55 60

Asp Cys Ile Be Cys Lys Tyr Gly Gln Asp Tyr Be Thr His Trp Asn65 70 75 80 -Asp Read Leu Phe Cys Read Arg Cys Thr Arg Cys Asp Be Gly Glu Vai85 90 95

Glu Read Be Pro Cys Thr Thr Thr Asn Thr Go Cys Gln Cys Glu100 105 110

Glu Gly Thr Phe Arg Glu Glu Asp Be Pro Glu Met Cys Arg Lys Cys115 120 125

Arg Gly Cys Pro Arg Gly Met Gys Lys Go Gly Asp Cys Thr Pro130 135 140

Trp Ser Asp lie Glu Cys Goes His Lys Glu Be Gly Thr Lys His Ser145 150 155 160

Gly Glu Wing Pro Wing Go Glu Glu Wing Go Thr Be Sero Gly Thr165 170 175

Pro Wing Ser Pro Cys Ser Leu Ser180

<210> 596 <211> 17 <212> PRT <213> Artificial

<220>

<223> synthetic CDRH3 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y, S, R, P, or G

<220>

<221> MISC_FEATURE

<222> (8). . (8)

<223> X is R, Y, or S

<220>

<221> MISC_FEATURE

<222> (9) .. (9)

<223> X is G, Y, or S

<220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is S, Y, or R

<220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is G or A

<220>

<221> MISC FEATURE <222> (15) .. (15)

<223> X is L or F

<400> 596

Tyr Arg Xaa Tyr Arg Tyr Gly Xaa Xaa Xaa Gly Ser Tyr Xaa Xaa Asp1 5 10 15

Tyr

<210> 597

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S

<400> 597

Gln Gln Xaa Xaa Xaa Ser Pro Ser Thr1 5

<210> 598

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S

<400> 598Gly Phe Xaa Ile Xaa Ser Ser Ser Ile His1 5 10

<210> 599

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is independently Y or S <220>

<221> MISC_FEATURE

<222> (10) .. (10)

<223> X is independently Y or S

<400> 599

Xaa Ile Ser Pro Xaa Xaa Gly Tyr Thr Xaa Tyr Ala Asp Ser Lys Vai15 10 15

Gly

<210> 600

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Synthetic BDF1 CDRH1

<400> 600

Ile Gly Lys Ser Gly Ile His1 5

<210> 601

<211> 14

<212> PRT

<213> Artificial

<220> <223> Synthetic BDF1 CDRH2 <400> 601

Go Wing Go Ile Tyr Pro His Asp Gly Asn Thr Wing Tyr Ala1 5 10

<210> 602

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic BDF1 CDRH3 <400> 602

Arg Read Leu Read Le Go Arg Met Trp Met Asp1 5 10

<210> 603

<211> 16

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 603

Tyi Stbt Ser Tyr Tyr Ser

1 5

<210> 604

<211> 411

<212> PRT

<213> Homo sapiens

<400> 604

10 15

Met Glu Gln Arg Gly Gln Asn Wing Pro Wing Wing Be Gly Wing Arg Lys15 10 15

Arg His Gly Pro Gly Pro Arg Glu Wing Arg Gly Wing Arg Pro Gly Leu20 25 30

Arg Go Pro Lys Thr Leu Go Leu Go Go Ward Wing Go Leu Leu Leu

35 40 45

Will Be Glu Wing Be Wing Leu Ile Thr Gln Gln Asp Leu Wing Pro Gln

50 55 60

Gln Arg Wing Ala Pro Gln Gln Lys Arg Be Ser Pro Be Glu Gly Leu65 70 75 80

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser85 90 95Cys Lys Tyr

Cys Read Arg Cys Thr Arg Cys Asp

115 120

Thr His Trp Asn Asp Leu Leu Phe105 110

Be Gly Glu Go Glu Leu Be Pro125

Cys Thr Thr Thr Arg Asn Thr Go Cys Gln Cys Glu Glu Gly Thr Phe130 135 140

Arg Glu Glu Asp Be Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys145 150 155 160

Pro Arg Gly Met Goes Lys Goes Gly Asp Cys Thr Pro Trp Be Asp lie165 170 175

Glu Cys Go His Lys Glu Be Gly lie lie lie Gly Go Thr Ala180 185 190

Wing Go Go Leu lie Go Wing Go Phe Go Cys Lys Ser Leu Leu Trp195 200 205

Lys Lys Goes Read Pro Tyr. Read Lys Gly lie Cys Gly Gly Gly Gly Gly210 215 220

Asp Pro Glu Arg Go Asp Arg Be Ser Gln Arg Pro Gly Wing Glu Asp225 230 235 240

Asn Goes To Read Asn Goes To Go

Glu Gln Glu Met Glu Go Gln Glu Pro Wing Glu Pro Thr Gly Go Asn260 265 270

Met Leu Be Pro Gly Glu Be Glu His Leu Leu Glu Pro Wing Glu Ala275 280 285

Glu Arg Ser Gln Arg Arg Arg Read Leu Go To Wing Asn Glu Gly Asp290 295 300

Pro Thr Glu Thr Leu Arg Gln Cys Ashe Asp Ashe Ashe Phe Asp Ashe Leu Vai305 310 315 320

Pro Phe Asp Be Trp Glu Pro Read Met Arg Lys Read Gly Read Met Asp325 330 335

Asn Glu lie Lys Go Wing Lys Wing Glu Wing Wing Gly His Arg Asp Thr340 345 350

Leu Tyr Thr Met Leu lie Lys Trp Goes Asn Lys Thr Gly Arg Asp Ala355 360 365

Be Going His Thr Leu Asu Asp Wing Leu Glu Thr Leu Gly Glu Arg Leu370 375 380

Lys Wing Gln Lys lie Glu Asp His Read Leu Ser Ser Gly Lys Phe Met385 390 395 400Tyr Leu Glu Gly Asn Asp Wing Ser Ser Leu Ser405 410

<210> 605 <211> 120 <212> PRT <213> Mus musculus

<400> 605

Gly Leu Gln Arg Pro Glu Glu Be Pro Be Arg Gly Pro Cys Leu Wing 1 5 10 15

Gly Gln Tyr Leu 20 Be Glu Gly Asn Cys 25 Lys Pro Cys Arg Glu 30 Gly lie

Asp Tyr Thr 35 Be His Ser Asn His 40 Ser Leu Asp Ser Cys 45 Ile Leu Cys

Thr Go 50 Cys Lys Glu Asp Lys 55 Go Go Glu Thr Arg 60 Cys Asn lie Thr

.Thr Asn 65 Thr Go Cys Arg 70 Cys Lys Pro Gly Thr 75 Phe Glu Asp Lys Asp 80

Ser Pro Glu lie Cys 85 Gln Ser Cys Ser Asn 90 Cys Thr Asp Gly Glu 95 Glu

Glu Leu Thr Be 100 Cys Thr Pro Glu 105 Asn Arg Lys Cys Will 110 Be Lys

Thr Wing Trp 115 Wing Ser Trp His Lys 120

<210> <211> <212> <213> 606 281 PRT Homo sapiens

<400> 606

Met Ala 1 Met Met Glu 5 Goes Gln Gly Gly Pro 10 Get Read Gly Gln Thr 15 Cys

Go Leu lie Go 20 lie Phe Thr Go Leu 25 Leu Gln Ser Leu Cys 30 Go Ala

Go Thr Tyr 35 Go Tyr Phe Thr Asn 40 Glu Read Lys Gln Met 45 Gln Asp Lys

Tyr Ser Lys Ser Gly lie Cys Wing Phe Leu Lys Glu Asp Asp Ser Tyr

50 55 60

Trp Asp Pro Asn Asp Glu Glu Be Met Asn Be Pro Cys Trp Gln Go65 70 75 80Lys Trp Gln Read Le Arg Gln Read Le Arg Lys Met lie Leu Thr Thr85 90 95

Glu Glu Thr lie Ser Thr Go Gln Glu Lys Gln Gln Asn lie Ser Pro100 105 110

Leu Goes Arg Glu Arg Gly Pro Gln Arg Goes Ward Wing His lie Thr Gly115 120 125

Thr Arg Gly Arg Be Asn Thr Read Be Ser Pro Asn Ser Lys Asn Glu130 135 140

Lys Ala Read Gly Arg Lys lie Asn Be Trp Glu Be Be Arg Be Gly145 150 155 160

His Be Phe Leu Be Asn Leu His Leu Arg Asn Gly Glu Leu Go lie165 170 175

His Glu Lys Gly Phe Tyr Tyr lie Tyr Ser Gln Thr Tyr Phe Arg Phe180 185 190

Gln Glu Glu lie Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Go Gln195 200 205

Tyr lie Tyr Lys Tyr Thr Be Tyr Pro Asp Pro lie Leu Leu Met Lys210 215 220

Be Wing Arg Asn Be Cys Trp Be Lys Asp Wing Glu Tyr Gly Leu Tyr225 230 235 240

Ser -HL- and -Tyr- <3in- Giy-G-l-y-lie-Phe-Si-u-Leu-Lys-G-lu-Asn-Asp-Arg-245 250 255

Phe Will Be Go Thr Asn Glu His Leu lie Asp Met Asp His Glu Ala260 265 270

Be Phe Phe Gly Wing Phe Leu Go Gly275 280

<210> 607

<211> 168

<212> PRT

<213> Artificial

<220>

<223> Synthetic Apo-2L

<400> 607

Go Arg Glu Arg Gly Pro Gln Arg Go Ward Wing His lie Thr Gly Thr1 5 10 15

Arg Gly Arg Be Asn Thr Read Be Be Pro Asn Be Lys Asn Glu Lys20 25 30

Wing Leu Gly Arg Lys lie Asn Be Trp Glu Be Be Arg Be Gly His35 40 45Ser Phe Leu Be Asn Leu50

Glu Lys Gly Phe Tyr Tyr65 70

Glu Glu lie Lys Glu Asn85

lie Tyr Lys Tyr Thr Ser100

Arg Wing Asn Ser Cys Trp115

lie Tyr Gln Gly Gly lie130

Will Be Go Thr Asn Glu145 150

Phe Phe Gly Wing Phe Leu165

<210> 608 <211> 6 <212> PRT <213> Artificial

<220>

«2-2-3> —GBR-H-3 — s-i-n-feti

<220>

<221> MISC_FEATURE <222> (1) .. (3) <223> X is any amino acid except cysteine

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is any amino acid and may vary in length <220>

<221> MISC_FEATURE

<222> (5) .. (6)

<223> X is any amino acid except cysteine

<400> 608

Xaa Xaa Xaa Xaa Xaa Xaa1 5

<210> 609

<211> 9

<212> PRT

<213> Artificial

His Leu Arg Asn Gly Glu Leu Go lie His55 60

lie Tyr Ser Gln Thr Tyr Phe Arg Phe Gln75 80

Thr Lys Asn Asp Lys Gln Met Going Gln Tyr90 95

Tyr Pro Asp Pro lie Leu Leu Met Lys Ser105 110

Ser Lys Asp Wing Glu Tyr Gly Leu Tyr Ser120 125

Phe Glu Leu Lys Glu Asn Asp Arg lie Phe135 140

His Leu lie Asp Met Asp His Glu Ala Ser

155 160

Go Gly <220>

<223> Synthetic CDRL3 <220>

<221> MISC_FEATURE

<222> (1). . (5)

<223> X is S or any of Y, W, R, or F <220>

<221> misc_feature

<222> (6) .. (6)

<223> Xaa can be any naturally occurring amino acid <220>

<221> misc_feature

<222> (8) .. (8)

<223> Xaa can be any naturally occurring amino acid

<400> 609

Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr

<210> 610

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<220>

<221> MISC_FEATURE

<222> (1) .. (5)

<223> X is any amino acid except cysteine <220>

<221> misc_feature

<222> (6) .. (6)

<223> Xaa can be any naturally occurring amino acid <220>

<221> misc_feature

<222> (8) .. (8)

<223> Xaa can be any naturally occurring amino acid

<400> 610

Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1 5

<210> 611

<211> 19

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH3 <220>

<221> MISC_FEATURE <222> (1) .. (17)

<223> X is S or any of A, C, F, G, I, L, W, P, R, T, W, or Y, or not present

<220>

<221> MISC_FEATURE <222> (18) .. (18)

<223> X is G or A, or not present <220>

<221> MISC_FEATURE <222> (19) .. (19)

<223> X is F, L, I, or M, or not present <400> 611

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa15 10 15

Xaa Xaa Xaa

<210> 612

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (3). . (3)

<223> X is S or any of Y, W, R, or F <220>

<221> MISC_FEATURE

<222> (5) .. (8)

<223> X is S or any of Y, W, R, or F

<400> 612

Gly Phe Xaa lie Xaa Xaa Xaa Xaa lie His

1 5 10

<210> 613

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is S or any of Y, W, R, or F <220>

<221> MISC_FEATURE

<222> (3). . (3)

<223> X is S or any of Y, W, R, or F <220>

<221> MISC_FEATURE

<222> (5) .. (6)

<223> X is S or any of Y, W, R, or F <220>

<221> MISC_FEATURE

<222> (8) .. (8)

<223> X is S and one of Y, W, R, or F <220>

<221> MISC_FEATURE

<222> (10). . (10) ......

<223> X is S or any of Y, W, R, or F

<400> 613

Xaa Ile Xaa Pro Xaa Xaa Gly Xaa Thr Xaa Tyr Ala Asp Will Go Lys15 10 15

Gly

<210> 614

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <220>

<221> MISC_FEATURE

<222> (1). . (5)

<223> X is any amino acid except cysteine <220>

<221> MISC_FEATURE

<222> (6) .. (8)

<223> Xaa can be any naturally occurring amino acid

<400> 614

Gly Phe Xaa Ile Xaa Xaa Xaa Xaa Ile His15 10

<210> 615 <211> <212> <213> 17 Artificial PRT

<220> <223> synthetic CDRH2

<220> <221> <222> <223> MISC FEATURE (D • • (D X is any amino acid except cysteine

<220> <221> <222> <223> MISC FEATURE (3) .. (3) X is any amino acid except cysteine

<220> <221> <222>. <2.23> - MISC FEATURE (5). . (6). X. is._q.ual.q.u.er. amino acid., except for cis-thine

<220> <221> <222> <223> MI SC FEATURE (8). . (8) X is any amino acid except cysteine

<220> <221> <222> <2-23> - MISC FEATURE (10) .. (10) X is any amino acid except cysteine

<400> 615

Xaa lie Xaa Pro Xaa Xaa Gly Xaa 1 5 Thr Xaa 10 Tyr Ala Asp Ser

Gly <210> <211> <212> <213> 616 10 Artificial PRT

<220> <223> synthetic CDRH3

<220> <221> <222> <223> MI SC FEATURE (D. (5) X is any amino acid except cysteine

<220> <221> <222> <223> MISC FEATURE (6) .. (6) X is any amino acid except cysteine and may

15length

<220>

<221> MISC_FEATURE

<222> (7) .. (8)

<223> X is any amino acid except cysteine

<400> 616

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr15 10

<210> 617

<211> 21

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> X is Y, S, G, R, or E

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> X is Y, S, G, R, M, or A

<220> -

<221> MISC_FEATURE

<222> (3) .. (3)

<223> X is Y, S, G, R, W, or H <220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> X is Y, S, G, R, F, or Q <220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> X is G, Y, N, A, or S

<220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> X is F, M, L, A, R, G, H, W, V, Y, or S <220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> X is M, L, G, A, R, F, Y, or S, or is not present <220> <221> MISC_FEATURE <222> (8) .. (8)

<223> X is M, L, F, I, R, G, P, V, Y, or S, or not present

<220>

<221> MISC_FEATURE

<222> (9) .. (9)

<223> X is G, Y, R, or S, or not present

<220>

<221> MISC_FEATURE

<222> (10). . (10)

<223> X is M, F, G, Y, R, or S, or not present

<220>

<221> MISC_FEATURE

<222> (11) .. (11)

<223> X is A, G, Y, R, or S, or not present

<220>

<221> MISC FEATURE

<222> (12). . (12)

<223> X is T, M, _F _ / ._ A./....G.,_R.,_-T.^_Y-,_o.u_-S-,_jQlu., not_e.s….a_prjas „ then

<220>

<221> MISC_FEATURE

<222> (13). . (13)

<223> X is F, M, L, G, A, T, Y, or S, or not present

<220>

<221> MISC_FEATURE

<222> (14). . (14)

<223> X is L, F, M, I, G, A, T, or Y, or not present

<220>

<221> MISC_FEATURE

<222> (15) .. (15)

<223> X is M, Y, G, L, or R, or not present

<220>

<221> MISC_FEATURE

<222> (16) .. (16)

<223> X is Y or G, or not present <220>

<221> MISC_FEATURE

<222> (17). . (17)

<223> X is R, M, or G, or not present

<220>

<221> MISC_FEATURE

<222> (18). . (18)

<223> X is G or A

<220> <221>

MISC FEATURE <222> (19) .. (19)

<223> X is F, M, L, or I

<400> 617

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15

Xaa Xaa Xaa Asp Tyr20

<210> 618 <211> 48 <212> Artificial DNA <213>

<220>

<223> synthetic oligonucleotides <400> 618

gcagcttctg gcttctmtat ttmttmttmt tmtatacact gggtgcgt 48.

<210> 619

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 619 - .........

ctggaatggg ttgcatmtat ttmtccatmt tmtggttmta cttmttatgc cgatagcgtc 60

<210> 620

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 620

acttattact gtcagcaatm ttmttmttmt ccatmtacgt tcggacaggg tacc 54

<210> 621

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 621

gtctattatt gtgctcgckc tkctkctkct gstwtkgact actggggtca agga 54

<210> 622 <211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 622

gtctattatt gtgctcgckc tkctkctkct kctgstwtkg actactgggg tcaagga

<210> 623

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 623

gtctattatt gtgctcgckc tkctkctkct kctkctgstw tkgactactg gggtcaagga

<210> 624

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 624

gtctattatt gtgctcgckc tkctkctkct kctkctkctg stwtkgacta ctggggtcaa

gga

<210> 625

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 625

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctgstwtkga ctactggggt

caagga

<210> 626

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 626gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctgstwt kgactactgg 60

ggtcaagga 69

<210> 627

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 627

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctgs twtkgactac 60

tggggtcaag ga 72

<210> 628 <211> 75 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 628

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tgstwtkgac 60

tactggggtc aagga 75

<210> 629

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 629

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctgstwtk 60

gactactggg gtcaagga 78

<210> 630

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 630

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctkctgst 60

wtkgactact ggggtcaagg a

81 <210> 631

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 631

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctkctkct 60

gstwtkgact actggggtca agga 84

<210> 632

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 632

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctkctkct 60

kctgstwtkg actactgggg tcaagga 87

<210> 633

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 633

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctkctkct 60

kctkctgstw tkgactactg gggtcaagga

<210> 634

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides <400> 634

gtctattatt gtgctcgckc tkctkctkct kctkctkctk ctkctkctkc tkctkctkct 60

kctkctkctg stwtkgacta ctggggtcaa gga 93

<210> 635

<211> 54

<212> DNA

<213> Artificialgga

<210> 639

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 639

54

<220>

<223> synthetic oligonucleotides <400> 635

gtctattatt gtgctcgcts ctsctsctsc gstwtkgact actggggtca agga

<210> 636

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 636

gtctattatt gtgctcgcts ctsctsctsc tscgstwtkg actactgggg tcaagga 57

<210> 637

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 637

- gtctattatt gtgctcgcts ctsctsctsc tsctscgstw tkgactactg gggtcaagga 60

<210> 638

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 638

gtctattatt gtgctcgcts ctsctsctsc tsctsctscg stwtkgacta ctggggtcaa 60

63

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct scgstwtkga ctactggggt 60

caagga

66

<210> 640 <211> 69 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 640

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctscgstwt kgactactgg 60

ggtcaagga 69

<210> 641

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 641

gtctattatt.-gtgctcgcts- -ctsctsctsc.tsctsctsct sctsctscg.s._twtkg.actac ..... 6.0

tggggtcaag ga 72

<210> 642

<211> 75

<212> DNA

<213> Artificial

<220>

<223> - synthetic oligonucleotides <400> 642

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts cgstwtkgac 60

tactggggtc aagga 75

<210> 643

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 643

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctscgstwtk 60

gactactggg gtcaagga 78

<210> 644

<211> 81

<212> DNA

<213> Artificial

<220> <223> synthetic oligonucleotides <400> 644

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctsctscgst 60

wtkgactact ggggtcaagg a 81

<210> 645

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 645

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctsctsctsc 60

gstwtkgact actggggtca agga 84

<210> 646 <211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 646

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctsctsctsc 60

tscgstwtkg actactgggg tcaagga87

<210> 647 <211> 90 <212> Artificial DNA <213>

<220>

<223> synthetic oligonucleotides <400> 647

gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctsctsctsc 60

tsctscgstw tkgactactg gggtcaagga

<210> 648

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides

<400> 648gtctattatt gtgctcgcts ctsctsctsc tsctsctsct sctsctscts ctsctsctsc 60

tsctsctscg stwtkgacta ctggggtcaa gga 93

<210> 649

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 649

gtctattatt gtgctcgcty ctyctyctyc gstwtkgact actggggtca agga 54

<210> 650 <211> 57 <212> Artificial DNA <213>

<220>

<223> synthetic oligonucleotides

<400> 650

gtctattatt gtgctcgcty ctyctyctyc tycgstwtkg actactgggg tcaagga 57

<210> 651

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 651

gtctattatt gtgctcgcty ctyctyctyc tyctycgstw tkgactactg gggtcaagga 60

<210> 652

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 652

gtctattatt gtgctcgcty ctyctyctyc tyctyctycg stwtkgacta ctggggtcaa 60

gga

<210> 653

<211> 66

<212> DNA

<213> Artificial

63

<220> <223> synthetic oligonucleotides <400> 653

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct ycgstwtkga ctactggggt 60

caagga 66

<210> 654

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 654

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctycgstwt kgactactgg 60

ggtcaagga 69

<210> 655

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 655

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycgs twtkgactac 60

tggggtcaag ga 72

<210> 656

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 656

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty cgstwtkgac 60

tactggggtc aagga 75

<210> 657

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 657

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctycgstwtk

60gactactggg gtcaagga

<210> 658

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 658

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctyctycgst 60

wtkgactact ggggtcaagg a 81

<210> 659 <211> 84 <212> Artificial DNA <213>

<220> <223> synthetic oligonucleotides

<400> 659

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctyctyctyc 60

gstwtkgact actggggtca agga 84

<210> 660

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 660

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctyctyctyc 60

tycgstwtkg actactgggg tcaagga 87

<210> 661

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 661

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctyctyctyc 60

tyctycgstw tkgactactg gggtcaagga

<210> 662 <211> 93

90 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 662

gtctattatt gtgctcgcty ctyctyctyc tyctyctyct yctyctycty ctyctyctyc 60tyctyctycg stwtkgacta ctggggtcaa gga 93

<210> 663

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 663

gtctattatt gtgctcgcrg- crgcrgcrgc gstwtkgact <210> 664

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 664

gtctattatt gtgctcgcrg crgcrgcrgc rgcgstwtkg <210> 665

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 665

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcgstw <210> 666

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 666

.54

57

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcg stwtkgacta ctggggtcaa 60

gga

63 <210> 667

<211> 66

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 667

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcgstwtkga ctactggggt 60

caagga 66

<210> 668

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 668

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcgstwt kgactactgg 60

ggtcaagga 69

<210> 669

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 669

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcgs twtkgactac 60

tggggtcaag ga 72

<210> 670

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 670

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg cgstwtkgac 60

tactggggtc aagga 75

<210> 671

<211> 78

<212> DNA

<213> Artificial <220>

<223> synthetic oligonucleotides <400> 671

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcgstwtk 60

gactactggg gtcaagga 78

<210> 672

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 672

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcrgcgst 60wtkgactact ggggtcaagg a 81

<210> 673

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 673

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcrgcrgc 60

gstwtkgact actggggtca agga 84

<210> 674

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 674

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcrgcrgc 60

rgcgstwtkg actactgggg tcaagga 87

<210> 675

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 675gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcrgcrgc 60

rgcrgcgstw tkgactactg gggtcaagga 90

<210> 676

<211> 93

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 676

gtctattatt gtgctcgcrg crgcrgcrgc rgcrgcrgcr gcrgcrgcrg crgcrgcrgc 60

rgcrgcrgcg stwtkgacta ctggggtcaa gga 93

<210> 677

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 677

gtctattatt gtgctcgcak cakcakcakc gstwtkgact actggggtca agga 54

<210> 678

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 678

gtctattatt gtgctcgcak cakcakcakc akcgstwtkg actactgggg tcaagga 57

<210> 679

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 679

gtctattatt gtgctcgcak cakcakcakc akcakcgstw tkgactactg gggtcaagga 60

<210> 680

<211> 63

<212> DNA

<213> Artificial

<220> <223> synthetic oligonucleotides

<400> 680

gtctattatt gtgctcgcak cakcakcakc akcakcakcg stwtkgacta ctggggtcaa 60

gga 63

<210> 681

<211> 66

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 681

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcgstwtkga ctactggggt 60

caagga 66

<210> 682 <211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 682

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcakcgstwt kgactactgg 60ggtcaagga "~" '~~ "' 69

<210> 683

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 683

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcakcakcgs twtkgactac 60

tggggtcaag ga 72

<210> 684

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 684

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcakcakcak cgstwtkgac 60tactggggtc aagga 75

<210> 685

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 685

gtctattatt gtgctcgcak cakcakcakcgactactggg gtcaagga

akcakcakca kcakcakcak cakcgstwtk 60

78

<210> 686

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 686

gtctattatt gtgctcgcak cakcakcakcwtkgactact ggggtcaagg a

akcakcakca kcakcakcak cakcakcgst 60

81

<210> 687 <211> 84 <212> DNA <213> "Artificial"

<220>

<223> synthetic oligonucleotides

<400> 687

gtctattatt gtgctcgcak cakcakcakcgstwtkgact actggggtca agga

akcakcakca kcakcakcak cakcakcakc 60

84

<210> 688

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 688

gtctattatt gtgctcgcak cakcakcakcakcgstwtkg actactgggg tcaagga

akcakcakca kcakcakcak cakcakcakc 60

87

<210> 689 <211> 90 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 689

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcakcakcak cakcakcakc 60

akcakcgstw tkgactactg gggtcaagga

<210> 690

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides <400> 690

gtctattatt gtgctcgcak cakcakcakc akcakcakca kcakcakcak cakcakcakc 60

akcakcakcg stwtkgacta ctggggtcaa gga 93

<210> 691

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 691

gtctattatt gtgctcgcty atyatyatya gstwtkgact actggggtca agga 54

<210> 692

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 692

gtctattatt gtgctcgcty atyatyatya tyagstwtkg actactgggg tcaagga 57

<210> 693

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 693

gtctattatt gtgctcgcty atyatyatya tyatyagstw tkgactactg gggtcaagga 60 <210> 694

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 694

gtctattatt gtgctcgcty atyatyatya tyatyatyag stwtkgacta ctggggtcaa 60

63

gga

<210> 695

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 695

gtctattatt gtgctcgcty atyatyatya tyatyatyat yagstwtkga ctactggggt 60

66

caagga

<210> 696

<211> 69

<212> DNA

<213> Artificial

<220> <223> oligonucleotides

<400> 696

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyagstwt kgactactgg 60

ggtcaagga 69

<210> 697

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 697

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyags twtkgactac 60

tggggtcaag ga 72

<210> 698

<211> 75

<212> DNA

<213> Artificial <220>

<223> synthetic oligonucleotides <400> 698

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty agstwtkgac 60

tactggggtc aagga 75

<210> 699

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 699

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyagstwtk 60

gactactggg gtcaagga 78

<210> 700

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 700

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyatyagst 60

wtkgactact ggggtcaagg a 81

<210> 701

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 701

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyatyatya 60

gstwtkgact actggggtca agga 84

<210> 702

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 702gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyatyatya 60

tyagstwtkg actactgggg tcaagga 87

<210> 703

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 703

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyatyatya 60

tyatyagstw tkgactactg gggtcaagga

<210> 704

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides <400> 704

gtctattatt gtgctcgcty atyatyatya tyatyatyat yatyatyaty atyatyatya 60

tyatyatyag stwtkgacta ctggggtcaa gga 93

<210> 705

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 705

gtctattatt gtgctcgcar carcarcarc gstwtkgact actggggtca agga 54

<210> 706

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 706

gtctattatt gtgctcgcar carcarcarc arcgstwtkg actactgggg tcaagga 57

<210> 707

<211> 60

<212> DNA

<213> Artificial <220>

<223> synthetic oligonucleotides <400> 707

gtctattatt gtgctcgcar carcarcarc arcarcgstw tkgactactg gggtcaagga 60

<210> 708

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 708

gtctattatt gtgctcgcar carcarcarc arcarcarcg stwtkgacta ctggggtcaa 60

gga 63

<210> 709

<211> 66

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 709

gtctattatt gtgctcgcar carcarcarc arcarca rcgstwtkga ctactggggt 60

caagga 66

<210> 710

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 710

gtctattatt gtgctcgcar carcarcarc arcarca rcarcgstwt kgactactgg 60

ggtcaagga 69

<210> 711

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 711

gtctattatt gtgctcgcar carcarcarc arcarca rcarcarcgs twtkgactac 60tggggtcaag ga 72

<210> 712

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 712

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar cgstwtkgac 60

tactggggtc aagga 75

<210> 713

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 713

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcgstwtk 60

gactactggg gtcaagga 78

<210> 714 <211> 81 <212> DNA

<213> Artificial ------ ■ ----------- "

<220>

<223> synthetic oligonucleotides <400> 7Í4

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcarcgst 60

wtkgactact ggggtcaagg a 81

<210> 715

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 715

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcarcarc 60gstwtkgact actggggtca agga 84

<210> 716 <211> 87 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 716

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcarcarc 60

arcgstwtkg actactgggg tcaagga 87

<210> 717

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 717

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcarcarc 60

arcarcgstw tkgactactg gggtcaagga 90

<210> 718

<211> 93

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 718

gtctattatt gtgctcgcar carcarcarc arcarcar rcarcarcar carcarcarc 60

arcarcarcg stwtkgacta ctggggtcaa gga 93

<210> 719

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 719

gtctattatt gtgctcgcyc tyctyctyct gstwtkgact actggggtca agga 54

<210> 720

<211> 57

<212> DNA •

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 720

gtctattatt gtgctcgcyc tyctyctyct yctgstwtkg actactgggg tcaagga

<210> 721

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 721

gtctattatt gtgctcgcyc tyctyctyct yctyctgstw tkgactactg gggtcaagga

<210> 722

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 722

gtctattatt gtgctcgcyc tyctyctyct yctyctyctg stwtkgacta ctggggtcaa

gga

<210> 723

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 723

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctgstwtkga ctactggggt

caagga

<210> 724

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 724

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctgstwt kgactactggggtcaagga

<210> 725 <211> 72 <212> DNA <213> Artificial <220>

<223> synthetic oligonucleotides <400> 725

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctgs twtkgactac 60

tggggtcaag ga 72

<210> 726

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 726

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tgstwtkgac 60

tactggggtc aagga 75

<210> 727

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 727 ■ • • TT

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctgstwtk 60

gactactggg gtcaagga 78

<210> 728

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 728

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctyctgst 60

wtkgactact ggggtcaagg a 81

<210> 729

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 729

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctyctyct 60

gstwtkgact actggggtca agga 84

<210> 730

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 730

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctyctyct 60

yctgstwtkg actactgggg tcaagga 87

<210> 731

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 731

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctyctyct 60yctyctgstw tkgactactg gggtcaagga 90

<210> 732 "" '

<211> 93

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 732

gtctattatt gtgctcgcyc tyctyctyct yctyctycty ctyctyctyc tyctyctyct 60

yctyctyctg stwtkgacta ctggggtcaa gga 93

<210> 733

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 733

gtctattatt gtgctcgcmg cmgcmgcmgc gstwtkgact actggggtca agga 54

<210> 734 <211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 734

gtctattatt gtgctcgcmg cmgcmgcmgc mgcgstwtkg actactgggg tcaagga 57

<210> 735

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 735

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcgstw tkgactactg gggtcaagga 60

<210> 736

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 736

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcg stwtkgacta ctggggtcaa 60

gga

<210> 737

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 737

63

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcgstwtkga ctactggggt 60

caagga 66

<210> 738

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 738gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcmgcgstwt kgactactgg 60ggtcaagga 69

<210> 739

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 739

gtctattatt gtgctcgcmg cmgcmgcmgctggggtcaag ga

mgcmgcmgcm gcmgcmgcgs twtkgactac 60

72

<210> 740

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 740

gtctattatt gtgctcgcmg cmgcmgcmgctactggggtc aagga

mgcmgcmgcm gcmgcmgcmg cgstwtkgac 60

75

<210> 741

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 741

gtctattatt gtgctcgcmg cmgcmgcmgcgactactggg gtcaagga

mgcmgcmgcm gcmgcmgcmg cmgcgstwtk 60

78

<210> 742

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 742

gtctattatt gtgctcgcmg cmgcmgcmgcwtkgactact ggggtcaagg a

mgcmgcmgcm gcmgcmgcmg cmgcmgcgst 60

81 <210> 743

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 743

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcmgcmgcmg cmgcmgcmgc 60

gstwtkgact actggggtca agga 84

<210> 744

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 744

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcmgcmgcmg cmgcmgcmgc 60

mgcgstwtkg actactgggg tcaagga 87

<210> 745

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 745

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcmgcmgcmg cmgcmgcmgc 60

mgcmgcgstw tkgactactg gggtcaagga

<210> 746

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides <400> 746

gtctattatt gtgctcgcmg cmgcmgcmgc mgcmgcmgcm gcmgcmgcmg cmgcmgcmgc 60

mgcmgcmgcg stwtkgacta ctggggtcaa gga 93

<210> 747

<211> 54

<212> DNA

<213> Artificial <220>

<223> synthetic oligonucleotides <400> 747

gtctattatt gtgctcgcas shells gstwtkgact actggggtca agga

<210> 748

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 748

gtctattatt gtgctcgcas peel asccstwtkg actactgggg tcaagga 57

<210> 749

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 749

gtctattatt gtgctcgcas cascascasc ascascgstw tkgactactg gggtcaagga 60

<210> 750

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 750

gtctattatt gtgctcgcas cascascasc ascascascg stwtkgacta ctggggtcaa 60

63

gga

<210> 751

<211> 66

<212> DNA

<213> Artificial

<220>

<223> oligonucleotides

<400> 751

gtctattatt gtgctcgcas cascascasc ascascasca scgstwtkga ctactggggt 60caagga 66

<210> 752 <211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 752

gtctattatt gtgctcgcas rinds ascascasca scascgstwt kgactactgg 60

ggtcaagga 69

<210> 753

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 753

gtctattatt gtgctcgcas cascascasc ascascasca scascascgs twtkgactac 60

tggggtcaag ga ^ 2

<210> 754

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 754

gtctattatt gtgctcgcas rinds ascascasca scascascas cgstwtkgac 60

tactggggtc aagga 75

<210> 755

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 755

gtctattatt gtgctcgcas rinds ascascasca scascascas cascgstwtk 60

gactactggg gtcaagga 78

<210> 756

<211> 81

<212> DNA

<213> Artificial

<220> <223> synthetic oligonucleotides <400> 756

gtctattatt gtgctcgcas rinds ascascasca scascascas shellscgst 60

wtkgactact ggggtcaagg a 81

<210> 757

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 757

gtctattatt gtgctcgcas shells shells ascascasca scascascas shells 60

gstwtkgact actggggtca agga 84

<210> 758

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 758

gtctattatt gtgctcgcas shells shells ascascasca scascascas shells 60

ascgstwtkg actactgggg tcaagga 87

<210> 759

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 759

gtctattatt gtgctcgcas shells shells ascascasca scascascas shells 60

ascascgstw tkgactactg gggtcaagga

<210> 760

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides

<400> 760

gtctattatt gtgctcgcas peelscasc ascascasca scascascas shells 60cascascascg stwtkgacta ctggggtcaa gga 93

<210> 761

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 761

gtctattatt gtgctcgcts gtsgtsgtsg gstwtkgact actggggtca agga 54

<210> 762

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 762

gtctattatt gtgctcgcts gtsgtsgtsg tsggstwtkg actactgggg tcaagga 57

<210> 763

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 763

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsggstw tkgactactg gggtcaagga 60

<210> 764

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 764

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgg stwtkgacta ctggggtcaa 60

gga 63

<210> 765

<211> 66

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 765256

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sggstwtkga ctactggggt 60

caagga 66

<210> 766

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 766

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsggstwt kgactactgg 60

ggtcaagga 69

<210> 767

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 767

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsggs twtkgactac 60

tggggtcaag ga 72

<210> 768

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 768

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts ggstwtkgac 60

tactggggtc aagga 75

<210> 769

<211> 78

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 769

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsggstwtk 60gactactggg gtcaagga 78 <210> 770

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 770

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsgtsggst 60

wtkgactact ggggtcaagg a 81

<210> 771

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 771

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsgtsgtsg 60

gstwtkgact actggggtca agga 84

<210> 772

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 772

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsgtsgtsg 60

tsggstwtkg actactgggg tcaagga 87

<210> 773

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 773

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsgtsgtsg 60

tsgtsggstw tkgactactg gggtcaagga

<210> 774

<211> 93

<212> DNA

<213> Artificial

90 <220>

<223> synthetic oligonucleotides <400> 774

gtctattatt gtgctcgcts gtsgtsgtsg tsgtsgtsgt sgtsgtsgts gtsgtsgtsg 60

tsgtsgtsgg stwtkgacta ctggggtcaa gga 93

<210> 775

<211> 54

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 775

. gtctattatt gtgctcgctm ttmttmttmt gstwtkgact actggggtca agga 54

<210> 776

<211> 57

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 776

gtctattatt gtgctcgctm ttmttmttmt tmtgstwtkg actactgggg tcaagga 57

<210> 777

<211> 60

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 777

gtctattatt gtgctcgctm ttmttmttmt tmttmtgstw tkgactactg gggtcaagga 60

<210> 778

<211> 63

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 778

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtg stwtkgacta ctggggtcaa 60gga 63

<210> <211>

77966 <212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 779

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mtgstwtkga ctactggggt 60

caagga 66

<210> 780

<211> 69

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 780

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmtgstwt kgactactgg 60

ggtcaagga 69

<210> 781

<211> 72

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 781

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmtgs twtkgactac 60

tggggtcaag ga 72

<210> 782

<211> 75

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 782

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm tgstwtkgac 60

tactggggtc aagga 75

<210> 783

<211> 78

<212> DNA

<213> Artificial

<220> <223> synthetic oligonucleotides <400> 783

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmtgstwtk 60

gactactggg gtcaagga 78

<210> 784

<211> 81

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 784

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmttmtgst 60

wtkgactact ggggtcaagg a 81

<210> 785

<211> 84

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 785

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmttmttmt 60

gstwtkgact actggggtca agga 84

<210> 786

<211> 87

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides <400> 786

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmttmttmt 60

tmtgstwtkg actactgggg tcaagga 87

<210> 787

<211> 90

<212> DNA

<213> Artificial

<220>

<223> synthetic oligonucleotides

<400> 787

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmttmttmt

60tmttmtgstw tkgactactg gggtcaagga

<210> 788

<211> 93

<212> DNA

<213> Artificial

90

<220>

<223> synthetic oligonucleotides <400> 788

gtctattatt gtgctcgctm ttmttmttmt tmttmttmtt mttmttmttm ttmttmttmt 60

tmttmttmtg stwtkgacta ctggggtcaa gga 93

<210> 789

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 789

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 790

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 790

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 791

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 791

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 792 <211> 9 <212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 792

Gln Gln Be Tyr Tyr Be Pro Ser Thr

<210> 793

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 793

Gln Gln Be Tyr Tyr Be Pro Ser Thr

<210> 794

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 794

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr

<210> 795

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 795

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 796

<211> 9

<212> PRT

<213> Artificial

<220> <223> Synthetic CDRL3

<400> 796

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 797

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 797

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 798

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 798

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 799

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 799

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 800

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 800Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr

<210> 801

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 801

Gln Gln Tyr Tyr Tyr Pro Ser Thr

<210> 802

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3 <400> 802

Gln Gln Tyr Tyr Tyr

<210> 803

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 803

Gln Gln Be Tyr Tyr Be Pro Ser Thr

<210> 804

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 804

Gln Tyr Tyr Tyr Tyr Tyr Pro Ser Thr1 5

<210> 805 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 805

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 806

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 806

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 807

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 807

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 808

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 808

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 809

<211> 9

<212> PRT

<213> Artificial

<220> <223> Synthetic CDRL3

<400> 809

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 810

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 810

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 811

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 811

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 812

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 812

Gln Gln Tyr Being Tyr Tyr Pro Tyr Thr1 5

<210> 813

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 813Gln Gln Tyr Ser Be Tyr Pro Tyr Thr

<210> 814

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 814

Gln Gln Tyr Tyr Tyr Pro Tyr Thr

<210> 815

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 815

Gln Gln Ser Tyr Ser Ser Pro Tyr Thr

<210> 816

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 816

Gly Phe Tyr lie Ser Ser Ser Tyr lie His15 10

<210> 817

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 817

Gly Phe Tyr lie Ser Ser Ser Tyr lie His15 10

<210> 818 <211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 818

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 819

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 819

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His1 5 10

<210> 820

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 820

Gly Phe Ser Lie Ser Ser Ser Tyr lie His15 10

<210> 821

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 821

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 822

<211> 10

<212> PRT

<213> Artificial <220>

<223> synthetic CDRH1 <400> 822

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His1 5 10

<210> 823

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 823

Gly Phe Ser Ser Ser Ser Ser Tyr Lie His

10

<210> 824

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 824

Gly Phe Ser Tyr Tyr Tyr Ser Tyr lie His1 5 10

<210> 825

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 825

Gly Phe Ser Tyr Tyr Tyr Ser Tyr lie His1 5 10

<210> 826

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 826Gly Phe Ser Tyr Ser Ser Tyr lie

His10

<210> 827

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 827

Gly Phe Ser Tyr Ser Tyr Ser Tyr

His10

<210> 828

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 828

Gly Phe Ser Tyr Tyr

His10

<210> 829

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 829

Gly Phe Ser Tyr Tyr

His10

<210> 830

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 830

Gly Phe Ser Lie Ser Ser Ser Tyr lie His15 10

<210> 831 <211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 831

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His1 5 10

<210> 832

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 832

Gly Phe Tyr lie Tyr Ser Ser Tyr lie His15 10

<210> 833

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 833

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 834

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 834

Gly Phe Ser Lie Ser Ser Ser Tyr lie His15 10

<210> 835

<211> 10

<212> PRT

<213> Artificial <220>

<223> synthetic CDRH1 <400> 835

Gly Phe Ser lie Tyr Ser Ser Tyr lie His1 5 10

<210> 836

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 836

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 837

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 837

Gly Phe Tyr lie Tyr Ser Tyr lie His1 5 10

<210> 838

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 838

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 839

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRH1 <400> 839Gly Phe Ser Ile Ser Tyr Ser Ser Ile His1 5 10

<210> 840

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 840

Gly Phe Ser Ile Ser Tyr Ser Ser Ile His1 5 10

<210> 841

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 841

Gly Phe Ser Ile Ser Tyr Ser Ser Ile His1 5 10

<210> 842

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 842

Gly Phe Ser Ile Ser Ser Ser Ser Ile His15 10

<210> 843

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 843

Ser Ile Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly <210> 844

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 844

Be Ile Tyr Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 845

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 845

Be Ile Tyr Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 846

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 846

Be Ile Tyr Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Going Lys1 5 10 15

Gly

<210> 847

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 847See lie Tyr Pro Tyr Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys

1 5

Gly

<210> 848

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 848

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 849

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 849

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 850

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 850

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 851

<211> 17

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH2 <400> 851

Be Ile Tyr Pro Be Ser Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 852

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 852

Be Ile Tyr Pro Be Ser Gly Tyr Thr Ser Tyr Wing Asp Ser Will Lys1 5 10 15

Gly

<210> 853

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 853

Ser Ile Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 854

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 854

Ser Ile Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 855 <211> 17 <212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 855

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 856

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 856

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 857

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 857

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 858

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 858

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15Gly

<210> 859

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 859

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 860

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 860

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 861

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 861

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 862

<211> 17

<212> PRT

<213> Artificial

<220> <223>

Synthetic CDRH2 <400> 862

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 863

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 863

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 864

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 864

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 865

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 865

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 866

<211> 17

<212> PRT

<213> Artificial <220>

<223> synthetic CDRH2 <400> 866

Be Tyr Tyr Pro Tyr Gly Tyr Thr Tyr Tyr Ala Asp Be Going Lys1 5 10 15

Gly

<210> 867

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 867

Be Tyr Tyr Pro Tyr Gly Tyr Thr Tyr Tyr Ala Asp Be Going Lys1 5 10 15

Gly

<210> 868

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 868

Be Tyr Tyr Pro Tyr Gly Tyr Thr Be Tyr Ala Asp Be Go Lys1 5 10 15

Gly

<210> 869

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 869

Tyr lie Be Pro Be Gly Tyr Thr Be Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 870 <211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 870

Tyr Tyr Ser Tyr Ala Phe Asp Tyr

<210> 871

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 871

Tyr Tyr Ser Ser Gly Phe Asp Tyr

<210> 872

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 872

Tyr Tyr Ser Ser Gly Phe Asp Tyr

<210> 873

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 873

Tyr Tyr Ser Ser Gly Phe Asp Tyr1 5

<210> 874

<211> 8

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH3

<400> 874

Tyr Tyr Ser Tyr Wing Read Asp Tyr1 5

<210> 875

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 875

Trp Trp Ser Ser Ala Phe Asp Tyr1 5

<210> 876

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 876

Trp Trp Ser Ser Ala Met Asp Tyr1 5

<210> 877

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 877

Trp Trp Being Trp Gly Phe Asp Tyr1 5

<210> 878

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 878Trp Trp To Be Ala Phe Asp Tyr

<210> 879

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 879

Trp Trp Being Ser Gly Phe Asp Tyr

<210> 880

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 880

Trp Trp Ser Ser Ala Phe Asp Tyr

<210> 881

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 881

Trp Trp Ser Trp Wing Phe Asp Tyr

<210> 882

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 882

Trp Trp Ser Ser Ala Phe Asp Tyr1 5

<210> 883 <211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 883

Trp Trp Ser Ser Ala Phe Asp Tyr

<210> 884

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 884

Trp Trp Ser Be Wing Read Asp Tyr

<210> 885

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 885

Trp Trp Ser Ser Ala Phe Asp Tyr

<210> 886

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 886

Trp Trp Being Ser Gly Phe Asp Tyr1 5

<210> 887

<211> 8

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH3

<400> 887

Trp Trp Ser Ser Ala Phe Asp Tyr1 5

<210> 888

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 888

Trp Trp Ser Ser Ala Read Asp Tyr1 5

<210> 889

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 889

Trp Ser Ser Ser Gly Phe Asp Tyr1 5

<210> 890

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 890

Trp Trp Ser Ser Ala Met Asp Tyr1 5

<210> 891

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 891Trp Trp Ser Ser Ala Asp Tyr1 5

<210> 892

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 892

Trp Trp Ser Ser Ala Read Asp Tyr1 5

<210> 893

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 893

Phe Ser Phe Ser Ser Ser Ser Phe Ala Met Asp Tyr1 .5 10

<210> 894

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 894

Phe Ser Phe Ser Ser Ser Ser Phe Ala lie Asp Tyr1 5 10

<210> 895

<211> 13

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 895

Phe Phe Phe Phe Phe Phe Phe Phe Gly Phe Asp Tyr15 10

<210> 896 <211> 15

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 896

Tyr Tyr Be Tyr Tyr Be Tyr Tyr Tyr Be Phe Gly Read Asp Tyr1 5 10 15 <210> 897

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 897

Gln Gln Ser Trp Trp Trp Pro Trp Thr1 5

<210> 898

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 898

Gln Gln Be Trp Trp Trp Pro Be Thr1 5

<210> 899

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 899

Gln Gln Be Trp Trp Be Pro Ser Thr1 5

<210> 900

<211> 9

<212> PRT

<213> Artificial

<220> <223> Synthetic CDRL3

<400> 900

Gln Gln Trp Trp Trp Trp Pro Trp Thr1 5

<210> 901

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 901

Gln Gln Trp Trp Trp Trp Pro Ser Thr1 5

<210> 902

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 902

Gln Gln Be Trp Trp Trp Pro Be Thr1 5

<210> 903

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 903

Gln Gln Trp Trp Trp Trp Pro Ser Thr1 5

<210> 904

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 904Gln Gln Trp Trp Trp Trp Pro Ser Thr1 5

<210> 905

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 905

Gln Gln Trp Trp Trp Trp Pro Ser Thr1 5

<210> 906

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 906

Gln Gln Trp Be Trp Trp Pro Be Thr1 5

<210> 907

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 907

Gln Gln Trp Be Ser Trp Pro Ser Thr1 5

<210> 908

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 908

Gln Gln Trp Be Ser Trp Pro Ser Thr1 5

<210> 909 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 909

Gln Gln Ser Ser Ser Ser Ser Pro 5

<210> 910

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 910

Gln Gln Trp Ser Trp Ser Pro 1

<210> 911

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 911

Gln Gln Trp Be Ser Trp Pro Ser Thr1 5

<210> 912

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 912

Gln Gln Trp Be Ser Trp Pro Ser Thr1 5

<210> 913

<211> 9

<212> PRT

<213> Artificial

<220> <223> Synthetic CDRL3

<400> 913

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 914

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 914

Gln Gln Tyr Tyr Tyr Ser Pro Ser Thr1 5

<210> 915

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 915

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 916

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 916

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 917

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400>

917Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 918

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 918

Gln Gln Ser Tyr Tyr Tyr Pro Ser Thr1 5

<210> 919

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 919

Gln Gln Be Tyr Tyr Be Pro Ser Thr1 5

<210> 920

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 920

Gln Gln Ser Ser Ser Ser Ser Pro 5

<210> 921

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 921

Gln Gln Ser Tyr Ser Tyr Pro Tyr Thr1 5

<210> 922 <211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 922

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 923

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRL3

<400> 923

Gln Gln Tyr Tyr Ser Ser Pro Tyr Thr1 5

<210> 924

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 924

Gly Phe Ser Trp Trp Ser Trp Lie His15 10

<210> 925

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 925

Gly Phe Ser Trp Trp Ser Trp Lie His15 10

<210> 926

<211> 10

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH1 <400> 926

Gly Phe Ser lie Trp Trp Ser lie Trp His1 5 10

<210> 927

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 927

Gly Phe Ser lie Trp Trp Ser lie Trp His1 5 10

<210> 928

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 928

Gly Phe Ser lie Trp Ser Ser lie Trp His1 5 10

<210> 929

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 929

Gly Phe Ser lie Trp Trp Ser lie Trp His1 5 10

<210> 930

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 930Gly Phe Ser lie Ser Trp Ser Trp lie

His10

<210> 931

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 931

Gly Phe Ser Trp Trp

His10

<210> 932

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 932

Gly Phe Ser Trp Trp

His10

<210> 933

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 933

Gly Phe Ser Trp Trp

His10

<210> 934

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 934

Gly Phe Trp lie Trp Ser Ser Ser Ser His15 10

<210> 935 <211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 935

Gly Phe Trp lie Trp Ser Ser Ser Ser His15 10

<210> 936

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 936

Gly Phe Ser Ser Ser Ser Ser Ser Trp His1 5 10

<210> 937

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 937

Gly Phe Ser Ser Ser Ser Ser Ser Ser His 10

<210> 938

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 938

Gly Phe Trp lie Trp Ser Ser Ser Ser His15 10

<210> 939

<211> 10

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH1 <400> 939

Gly Phe Ser Lie Trp Ser Ser Lie His1 5 10

<210> 940

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 940

Gly Phe Ser Tyr Tyr Ser Tyr Tyr lie His1 5 10

<210> 941

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 941

Gly Phe Tyr lie Ser Tyr Ser Tyr lie His15 10

<210> 942

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 942

Gly Phe Tyr lie Ser Ser Ser Tyr lie His15 10

<210> 943

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 943Gly Phe Ser Ser Ser Ser Ser Tyr lie His1 5 10

<210> 944

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1

<400> 944

Gly Phe Ser Be Ser Ser Ser Ser Tyr lie His1 5 10

<210> 945

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 945

Gly Phe Ser Tyr Ser Tyr Ser Tyr lie His15 10

<210> 946

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 946

Gly Phe Tyr lie Ser Ser Ser Tyr lie His15 10

<210> 947

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRHl

<400> 947

Gly Phe Tyr lie Ser Tyr Ser Ser His15 10

<210> 948 <211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 948

Gly Phe Tyr lie Ser Ser Ser Ser Ser His 5 5 10

<210> 949

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 949

Gly Phe Ser lie Tyr Tyr Ser Lie His15 10

<210> 950

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH1 <400> 950

Gly Phe Ser lie Tyr Tyr Ser Lie His15 10

<210> 951

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 951

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys15 10 15

Gly

<210> 952

<211> 17

<212> PRT <213> Artificial <220>

<223> synthetic CDRH2 <400> 952

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 953

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 953

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 954

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 954

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 955

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 955

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly95617PRT

Artificial <220>

<223> synthetic CDRH2 <400> 956

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys15 10 15

Gly

<210> 957

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 957

Ser lie Trp Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys15 10 15

Gly

<210> 958

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 958

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys15 10 15

Gly

<210> 959

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 959

<210><211><212> <213>

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys15 10 15Gly

<210> 960

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 960

Ser lie Ser Pro Ser Ser Gly Trp Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 961

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 961

Trp lie Be Pro Be Be Gly Be Thr Trp Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 962

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 962

Trp lie Be Pro Be Be Gly Be Thr Trp Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 963

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 963Trp lie Trp Pro Be Gly Trp Thr Be Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 964

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 964

Trp lie Be Pro Be Trp Gly Be Thr Trp Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 965

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 965

Trp lie Be Pro Be Be Gly Be Thr Trp Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 966

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 966

Trp lie Be Pro Be Be Gly Be Thr Trp Tyr Wing Asp Be Go Lys15 10 15

Gly

<210> 967

<211> 17

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH2 <400> 967

Ser lie Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Ser Lys1 5 10 15

Gly

<210> 968

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 968

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 969

<211> 17

<212> PRT

<213> Artificial

<220>

<-2-2-3> —sin ^ é-tieG -——-

<400> 969

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 970

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 970

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 971 <211> 17 <212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 971

Ser Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys1 5 10 15

Gly

<210> 972

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 972

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 973 <211> 17

<-2-1-2-> -PRT ----------------

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 973

Ser lie Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala Asp Ser Will Lys15 10 15

Gly

<210> 974

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 974

Be lie Be Pro Tyr Be Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys15 10 15Gly

<210> 975

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 975

Tyr lie Be Pro Be Gly Tyr Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 976

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2

<400> 976

Be Tyr Tyr Pro Tyr Be Gly Ser Tyr Tyr Wing Asp Ser Go Lys1 5 10 15

Giy

<210> 977

<211> 17

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH2 <400> 977

Be lie Tyr Pro Be Ser Gly Be Thr Tyr Tyr Wing Asp Be Go Lys1 5 10 15

Gly

<210> 978

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 978Trp Trp Ser Ser Ala lie Asp Tyr

<210> 979

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 979

Trp Trp Ser Be Wing Read Asp Tyr

<210> 980

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 980

Trp Trp Ser Be Wing Read Asp Tyr

<210> 981

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 981

Trp Trp Being Ser Gly Met Asp Tyr

<210> 982

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 982

Trp Trp Ser Ser Ala Read Asp Tyr1 5

<210> 983 <211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 983

Trp Trp Ser Ser Ala Phe Asp Tyr

<210> 984

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 984

Trp Trp Ser Be Wing Read Asp Tyr

<210> 985

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 985

Trp Ser Ser Ser Ala Phe Asp Tyr

<210> 986

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 986

Trp Trp Ser Ser Ala Met Asp Tyr1 5

<210> 987

<211> 8

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH3 <400> 987

Trp Ser Ser Trp Gly Leu Asp Tyr1 5

<210> 988

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 988

Be Ser Trp Ser Ser Trp Ser Ser Ala Asu Tyr1 5 10

<210> 989

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 989

Be Ser Trp Ser Ser Trp Ser Ser Ala Asp Tyr15 10

<210> 990

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 990

Ser Ser Ser Ser Ser Trp Trp Ser Ala lie Asp Tyr15 10

<210> 991

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRH3 <400> 991Ser Ser Trp Ser Ser Trp Ser Trp Wing Read Asp Tyr1 5 10

<210> 992

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 992

Be Ser Trp Ser Ser Trp Ser Ser Ala Met Asp Tyr1 5 10

<210> 993

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 993

Be Ser Trp Ser Ser Trp Ser Ser Ala Met Asp Tyr1 5 10

<210> 994

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 994

Tyr Tyr Ser Tyr Wing Read Asp Tyr1 5

<210> 995

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 995

Tyr Tyr Ser Tyr Ala Phe Asp Tyr1 5

<210> 996 <211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 996

Tyr Tyr Ser Tyr Ala Phe Asp Tyr

<210> 997

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 997

Tyr Tyr Ser Ser Ala Phe Asp Tyr

<210> 998

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 998

Tyr Tyr Ser Tyr Wing Read Asp Tyr

<210> 999

<211> 8

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3

<400> 999

Tyr Tyr Ser Tyr Ala Phe Asp Tyr1 5

<210> 1000

<211> 8

<212> PRT

<213> Artificial

<220> <223> synthetic CDRH3 <400> 1000

Tyr Tyr Ser Tyr Wing Read Asp Tyr1 5

<210> 1001

<211> 14

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 1001

Be Ser Tyr Ser Tyr Tyr Tyr Tyr Ser Tyr Wing Read Asp Tyr1 5 10

<210> 1002

<211> 15

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 1002

Tyr Tyr Be Ser Tyr Tyr Ser Tyr Ser Tyr Ser Tyr Ala Read Asp Tyr1 5 10 15

<210> 1003

<211> 18

<212> PRT

<213> Artificial

<220>

<223> synthetic CDRH3 <400> 1003

Tyr Tyr Be Ser Tyr Ser Ser Ser Ser Tyr Tyr Tyr Tyr Ala Phe1 5 10 15

Asp Tyr

<210> 1004

<211> 19

<212> PRT

<213> Artificial

<220>

<223> Synthetic CDRH3 <400> 1004Tyr Tyr Ser Ser Ser Ser Ser Tyr Ser Ser Ser Tyr Tyr Tyr Ala1 5 10 15

Phe Asp Tyr

<210> 1005

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides <400> 1005

gcagcttctg gcttctycat ttyctyctyc tycatacact gggtgcgt 48

<210> 1006

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides <400> 1006

ctggaatggg ttgcatycat ttycccatyc tycggttyca cttyctatgc cgatagcgtc 60

<210> 1007

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides <400> 1007

acttattact gtcagcaaty ctyctyctyc ccatycacgt tcggacaggg tacc 54

<210> 1008

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides <400> 1008

gcagcttctg gcttcmgcat tmgcmgcmgc mgcatacact gggtgcgt 48

<210> 1009

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides <400> 1009

ctggaatggg ttgcamgcat tmgcccamgc mgcggtmgca ctmgctatgc cgatagcgtc 60

<210> 1010

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides

<400> 1010

acttattact gtcagcaamg cmgcmgcmgc ccamgcacgt tcggacaggg tacc 54

<210> 1011

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides

<400> 1011

gcàgcttctg gcttctsgat ttsgtsgtsg tsgatacact gggtgcgt 48

<210> 1012

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides

<400> 1012

ctggaatggg ttgcatsgat ttsgccatsg tsgggttsga cttsgtatgc cgatagcgtc 60

<210> 1013

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Synthetic oligonucleotides

<400> 1013

acttattact gtcagcaats gtsgtsgtsg ccatsgacgt tcggacaggg tacc 54

Claims (117)

1. Polypeptide, which comprises an immunoglobin heavy chain variable domain in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22) where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from S and Y; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), which X1 is position-50, according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; in which X5 is selected from Y and S, and in which X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X.14-X15-X16-DY (SEQ ID NO: ED: -31), wherein X1 is position 95 according to the Kabat numbering system, and wherein X1 is selected from R, Y, M; X 2 is selected from Y and R; X3 is selected from Y, S, R, P and G, X4 is selected from Y and S; X5 is selected from Y, S, R and H; X6 is selected from R, Y and S; X7 is selected from G, Y and S; X8 is selected from R, Y and S; X9 is selected from G, Y and S; X10 is selected from R, Y and S; X11 is selected from G, Y and S; X12 is selected from S, Y, R, G and A. X13 is selected from G and Y; X 14 is selected from L, M, R, G and A; and X15 is selected from G, F and L or not present, and X16 is F or not present. 2. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), where X1 is the -50 position according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; in which X5 is selected from Y and S, and in which X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ ID NO ED: 24), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio. 50% Y, 25% S, and 25% G; characterized in that the amino acid pellets at each of the positions of X7-X17 are selected from an amino acid pool at a molar ratio of 50% Y, 25% S and 25% G, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F. 3. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 28 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), where X1 is the -50 position according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; in which X5 is selected from Y and S, and in which X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ ID NO ED: 26), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the X1-X6 positions are selected from an amino acid pool in a molar ratio. 25% Y, 50% S, and 25% R; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 25% Y, 50% S and 25% R, or not present; wherein X18 is selected from G and A; where X19 is selected from I, M, L and F. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), where X1 is the -50 position according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; in which X5 is selected from Y and S, and in which X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ ID NO ED: 27), wherein X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the X1-X6 positions are selected from an amino acid pool in a molar ratio. 38% Y, 25% S, and 25% G and 12% R, characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool at a molar ratio of -38. % Y, 25% S, and 25% G and 12% R, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), which, X1 is position-50, according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; in which X5 is selected from Y and S, and in which X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY (SEQ ID NO ED: 28), where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from an amino acid pool at a molar ratio. 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I , 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 20% Y, 26% S, -26% G, 13% R, 1% A , 1% D, 1% E, 1% F, 1% H, 1% I, -1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V and -1% W, or not present; where X18 is selected from G and A, and in which X19 is selected from I, M, L and F. Polypeptide according to one of claims 1 to 5, characterized in that the CDRH1 comprises an amino acid sequence selected from any of SEQ ID Nos: 524-540 and 189-294. Polypeptide according to one of Claims 1 to 5, characterized in that the CDRH2 comprises an amino acid sequence selected from any of SEQ ID Nos: 541-557 and 295-400. Polypeptide according to one of claims 1 to 5, characterized in that the CDRH3 comprises a selected amino acid sequence from any of SEQ ID Nos: 558-574 and 401-506. Polypeptide according to claim 1, characterized in that the position of amino acid X1 in CDRH3 is position 95e and is selected from R and Y; X3 is position 97 and is S; X8 is amino acid position 100b and is S; X9 is the position of amino acid 100c and is Y; X10 and is apposition of amino acid 100d and is Y or R. Polypeptide according to Claim 1, characterized in that the CRDH3 position has the amino acid sequence X1-R-S-Y-R-Y-G-Y-X10-G-S-Y-X14-F-D-Y (SEQ ID NO: 575). Polypeptide according to one of Claims 1 to 10, characterized in that the polypeptide binds to human DR5. Polypeptide according to claim 11, characterized in that the polypeptide is an antibody. Polypeptide according to claim 12, characterized in that the heavy chain variable domain comprises: i) a CDRH1 containing an amino acid sequence GFYISSSSIH (SEQ ID NO: 576), ii) a CDRH2 containing an amino acid sequenceSISPSSGSTYYADSVKG (SEQ NO ID: 577); and iiii) a CDRH3 containing an amino acid sequence YRSYRYGSYYGSYGFDY (SEQ NO ID: 578). Polypeptide according to claim 12, characterized in that the variable chain heavy domain comprises: i) a CDRH1 containing an amino acid sequence GFYISSSSIH (SEQ ID NO: 579), ii) a CDRH2 containing an amino acid sequenceSISPSSGYTSYADSVKG ( SEQ ID NO: 580); eiii) a CDRH3 containing an amino acid sequenceRRSYRYGSYRGSYAFDY (SEQ NO ID: 581). Polypeptide according to one of claims 1 to 14, characterized in that the polypeptide binds to human and murine DR5. A polypeptide according to any one of claims 1 to 15, further comprising a light chain variable domain, wherein: (i) CDRL3 comprises an amino acid sequence QQ-X1-X2-X3-X4-P-X5- T (SEQ ID NO: 25); where X1 is position 91 and is selected from Y, H and S; X 2 is selected from Y and S; X3 is selected from Y, If T; X4 is selected from Y, S and T, and X5 is selected from S, P and Y. Polypeptide according to claim 16, further comprising a CDRL1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 29). Polypeptide according to claim 16, characterized in that it further comprises a CDRL2 having the amino acid sequence SASSLYS (SEQ ID NO: 30). 19. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where X1 is position 28 according to Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; (Ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), in which X1 is position-50 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y, and X6 is selected from S and Y; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-DY (SEQ ID NO: 582), wherein, X1 is amino acid position 95 according to the numbering system from Kabat and selected from S and Y; X 2 is selected from S and Y and R; X3 is selected from S, G, Y and H; X4 is selected from S, G, Y and R; X5 is selected from G and A; X6 is selected from F, M, L and A; and X7 is selected from F, M and L or is missing. Polypeptide according to claim 19, characterized in that the CDRH1 comprises an amino acid sequence selected from SEQ ID Nos: 189-198. Polypeptide according to claim 19, characterized in that the CDRH2 comprises an amino acid sequence selected from SEQ ID Nos: 295-304. Polypeptide according to Claim 19, characterized in that the CDRH3 comprises an amino acid sequence selected from SEQ ID Nos: 401-410. 23. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where X1 is position 28 according to Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; (Ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 22), in which X1 is position-50 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y, and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X7-X8-DY (SEQ ID NO: 583), wherein X1 is the amino acid position 95 according to the Kabat numbering system and selected from Y, S and G; X 2 is selected from Y, S, G, R and A; X3 is selected from G, Y, S and R; X4 is selected from G, Y and F; X5 is selected from Y, S, N and G X6 is selected from Y, R, H and W; and X7 is selected from G and A; and X8 is selected from F, M, L and I. Polypeptide according to claim 23, characterized in that the CDRH1 comprises an amino acid sequence selected from SEQ ID Nos: 199-216. Polypeptide according to claim 23, characterized in that the CDRH2 comprises an amino acid sequence selected from SEQ ID Nos: 305-322. Polypeptide according to Claim 23, characterized in that the CDRH3 comprises an amino acid sequence selected from SEQ ID Nos: 411-428. 27. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), where X1 is position 28 according to Kabat numbering system and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; (Ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG (SEQ ID NO: 23), in which X1 is position-50 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y, and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13-X14-X15-X16 -X17 -X18-X19-DY (SEQ ID NO: 584), wherein X1 is the position of amino acid 95 is selected from Y, S, R and G; X 2 is selected from Y, S, R and G; X3 is selected from S, Y, G and W; X4 is selected from S, Y, G and Q; X5 is selected from G, Y and S; X6 is selected from G, Y S, R and V; X7 is selected from S, Y, G and R; X8 is selected from Y, S, G, R, P and V; X9 is selected from G, A, Y, S and R; X10 is selected from M, F, G, Y, S and R; X11 is selected from A, Y, S, G and R; X12 is selected from I, M, F, L, A, G, S, Y, R and T; X13 is selected from F, M, L, G, A, Y, T and S or is not present; X 14 is selected from L, M, F, I, G, Y, A and T, and X15 is selected from M, L, Y, G and R or is not present; X16 is selected from Y and G or is not present; X17 is selected from R, M and G is not present; X18 is selected from P and A or is not present; and X18 is Lou is not present. 28. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH (SEQ ID NO: 22), characterized by the fact that G is position and X1 is position 28 according to the Kabat numbering system; and wherein X1-X5 are naturally present amino acids other than cysteine: (ii) CDRH2 comprises an amino acid sequence: X6-I-X7-P-X8-X9-G-X10-T-X11-YADSVKG, in which X6 is position 50 according to the Kabat numbering system, wherein X6-X11 are naturally present amino acids other than cysteine; and (iii) CDRH3 comprises an amino acid sequence: X12-X13-X14-X15-X16- (X17) n-X18-X19-D-Y, wherein X12 is position 95 according to the Kabat numbering system; and wherein n is an appropriate number which will maintain the functional activity of the heavy chain variable domain; and noqual, X12-X19 are naturally present amino acids other than cysteine. Polypeptide according to claim 28, characterized in that n is a number from 1 to 12. Polypeptide according to claim 28, characterized in that X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; and X6 is selected from Y and S. Polypeptide according to claim 28, characterized in that X 6 is selected from Y and S; X7 is selected from Y and S; X8 is selected from Y and S; X9 is selected from Y and S; X10 is selected from Y and S; and X11 is selected from Y and S. Polypeptide according to claim 28, characterized in that the amino acids at each of the X12-X17 positions are selected from an amino acid pool in a molar ratio of 50% Y, 25% S and 25% G, and X18 is selected from G and A, and X19 is selected from I, M, L, and F. Polypeptide according to claim 28, characterized in that the amino acids at each of the X12-X17 positions are selected from an amino acid pool in a molar ratio of 25% Y, 50% S and 25% R, where X18 is selected from G and A, and X19 is selected from I, M, L, and F. Polypeptide according to claim 28, characterized in that the amino acids at each of the X12-X17 positions are selected from an amino acid pool in a molar ratio of 38% Y, 25% S, 25% G is 12% of R, where X18 is selected from G and A, and X19 is selected from I, M, L, and F. A polypeptide according to claim 28, characterized in that the amino acids at each of the X12-X17 positions are selected from an amino acid pool in a molar ratio of 20% Y, 26% S, 26% G, 13 % R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, -1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; wherein X18 is selected from G and A; and X19 is selected from I, M, L, and F. Polypeptide according to one of claims 27-35, characterized in that the CDRH1 comprises an amino acid sequence selected from any of SEQ ID Nos: 217-294. Polypeptide according to one of claims 27-35, characterized in that the CDRH2 comprises an amino acid sequence selected from any of SEQ ID Nos: 323-400. Polypeptide according to one of claims 27-35, characterized in that the CDRH3 comprises an amino acid sequence selected from any of SEQ ID Nos: 429-506. 39. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, in which: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is apposition 50 according to the Kabat numbering system; where X1 is selected from Y and S; wherein X2 is selected from Y and S; where X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S; and wherein, X 6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 by the fact that X1 is position 95 according to the Kabat numbering system, and in which the amino acids at each of the positions of X1-X17 are selected from A, C, F, G, I, L, N, P, R, T, W or Y, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M. Polypeptide according to Claim 39, characterized in that the CDRH1 comprises an amino acid sequence selected from SEQ ID Nos: 816-842. Polypeptide according to Claim 39, characterized in that the CDRH2 comprises an amino acid sequence selected from SEQ ID Nos: 843-869. Polypeptide according to Claim 39, characterized in that the CDRH3 comprises an amino acid sequence selected from SEQ ID Nos: 870-896. 43. Polypeptide, which comprises an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; and wherein the amino acids at each of the positions of X1-X5 are selected from S and umd between F, L, I and M; and (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is apposition 50 according to the numbering system of Kabat; and wherein the amino acids at each of the positions of X1-X6 are selected from S and umd between F, L, I and M; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 by the fact that X1 is position 95 according to the Kabat numbering system, and the amino acids at each of the positions of X1-X19 are selected from S and one of Y, W, R or F, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M. Polypeptide according to Claim 43, characterized in that the CDRH1 comprises an amino acid sequence selected from SEQ ID Nos: 924-950. Polypeptide according to Claim 43, characterized in that the CDRH2 comprises an amino acid sequence selected from SEQ ID Nos: 951-977. Polypeptide according to Claim 43, characterized in that the CDRH3 comprises an amino acid sequence selected from SEQ ID Nos: 978-1004. Polypeptide according to one of Claims 2 to 8 or 16 to 46, characterized in that the polypeptide binds to human HER-2. Polypeptide according to claim 47, characterized in that the polypeptide is an antibody. Polypeptide according to claim 39 or 40, characterized in that the heavy chain variable domain comprises: -i) a CDRH1 containing an amino acid sequence GFSIYSSYIH (SEQ ID NO: 821), - ii) a CDRH2 containing a amino acid sequenceSIYPYSGYTSYADSVKG (SEQ ID No: 848); and iii) a CDRH3 containing an amino acid sequence WWSSAFDY (SEQ ID No: 875). A polypeptide according to claim 39 or 40, characterized in that the heavy chain variable domain comprises: -i) a CDRH1 containing an amino acid sequence GFSIWWSWIH (SEQ ID NO: 932), - ii) a CDRH2 containing a amino acid sequenceSISPSSGWTSYADSVKG (SEQ ID No: 959); and iii) a CDRH3 containing an amino acid sequence WWSSAMDY (SEQ ID No: 986). Polypeptide according to claim 39 or 40, characterized in that the heavy chain variable domain comprises: i) a CDRH1 containing an amino acid sequence (GFSISSSYIH (SEQ ID NO: 944), ii) a CDRH2 containing a sequence of amino acidsSIYPYSGYTSYADSVKG (SEQ ID No: 971); and (iii) a CDRH3 containing a YYSYALDY amino acid sequence (SEQ ID No: 998). A polypeptide according to claim 39 or 40, characterized in that the heavy chain variable domain comprises: i) a CDRH1 containing an amino acid sequence GFYISSSSIH (SEQ ID NO: 230), ii) a CDRH2 containing a sequence of amino acids YIYPSSGYTSYADSVKG (SEQ ID No: 336); eiii) a CDRH3 containing a GYYYSYYSGYALDY amino acid sequence (SEQ ID No: 442). A polypeptide according to one of claims 19-52, further comprising a light chain variable domain, wherein: (i) CDRL3 comprises an amino acid sequence QQ-X1-X2-X3-X4-P -X5-T (SEQ ID NO: 25); characterized by the fact that Xt is apposition 91 according to the Kabat numbering system and is selected from S and Y; X 2 is selected from S, Y and F; X3 is selected from Y; X4 is selected from Y and S, and X5 is selected from S and Y. 54. Polypeptide, which comprises a light chain variable domain having a CDRL3 sequence where CDRL3 has the amino acid sequence QQ-X1-X2-X3-X4-P-X5-T (SEQ ID NO: 25), where X1 is the position 91 according to Kabat numbering and is selected from Se Y; X 2 is selected from S, Y and F; X3 is selected from Y, S, F; X4 is selected from Y and S; X5 is selected from S and Y. 55. Polypeptide, which comprises an immunoglobulin light chain variable domain, wherein CDRL3 comprises a Q-Q-X1-X2-X3-X4-P-X5-T amino acid sequence; where X1 is position 91 according to the Kabat numbering system and the amino acids in each of the X1-X5 positions are selected from S and one of Y, W, R or F. 56. Polypeptide, which comprises an immunoglobulin light chain variable domain, in which: (i) CDRL1 comprises a first hypervariable consensus sequence or variant thereof containing a substitution in one or more positions, as compared to the corresponding hypervariable consensus sequence; a second hypervariable consensus sequence or variant thereof containing a substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and (iii) CDRL3 comprises an amino acid sequence: QQ-X1-X2-X3- (X4) n-X5-X6 -T, characterized by the fact that X1 is position 91 according to the Kabat numbering system. Polypeptide according to claim 56, characterized in that X1 is position 91 according to the Kabat numbering system and X1 is selected from Y and S; X2 is selected from Y and S X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Pe L; and X6 is selected from F, L, I and V. Polypeptide according to claim 56, characterized in that n is a number from 1 to 3. Polypeptide according to one of Claims 54 to 58, characterized in that the CDRL3 comprises an amino acid sequence selected from SEQ ID Nos: 83-188, 789-815 and 897-923. Polypeptide according to any one of claims 54 to 58, characterized in that it further includes a CDRL1 comprising an amino acid sequence RASQDVNTAVA (SEQ ID NO: 29). A polypeptide according to any one of claims 54 to 58 and claims 55 or 56, further comprising a CDRL2 comprising an amino acid sequence SASSLYS (SEQ ID NO: 30). ANTIBODY comprising a polypeptide having an immunoglobulin heavy chain variable domain according to one of claims 1 to 53, and a polypeptide having an immunoglobulin light chain variable domain according to one of claims 54 to 61. A polypeptide comprising at least two antibody variable domains comprising: (a) an antibody heavy chain variable domain comprising the polypeptide of one of claims 1 to 53; and (b) an antibody light chain variable domain comprising the polypeptide of one of claims 54 to 61. Polypeptide according to one of claims 1 to 63, characterized in that the polypeptide is a single chain Fv. Polypeptide according to one of Claims 1 to 63, characterized in that the polypeptide is a Fab polypeptide. Polypeptide according to one of Claims 1 to 58, characterized in that the polypeptide is a fully human polypeptide. A polypeptide according to any one of claims 1 to 66, further comprising the support regions FR1, FR2, FR3, and / or FR4 for a variable domain polypeptide corresponding to the CDRH1, CDRH2, CDRH3, and / or CDRL3 variant. , where the support regions obtained from a single model polypeptide. Polypeptide according to Claim 67, characterized in that each of the support regions comprises an amino acid sequence corresponding to the support region of the 4D5 polypeptide amino acid sequences (SEQ ID NO: 1 and 2). 69. ANTIBODY according to claim 67, characterized in that the antibody is in the support region corresponding to the amino acid sequence of the 4D5 antibody variant support region (SEQ ID NO: 14-17 and 18-21). Polypeptide according to one of Claims 1 to 69, characterized in that the polypeptide is additionally of a dimerization domain linked to the C-terminal region of the heavy chain variable domain. Polypeptide according to claim 70, characterized in that the dimerization domain contains a leucine zipper domain or a sequence having at least one cysteine residue. Polypeptide according to claim 70, characterized in that the dimerization domain comprises a hinge region of a polypeptide and a leucine zipper. Polypeptide according to claim 70, characterized in that the dimerization domain is a single cysteine. A fusion polypeptide comprising a polypeptide according to one of claims 1 to 73, characterized in that the variable domain polypeptide is fused to at least a portion of a viral capsid protein. 75. Fusion polypeptide according to claim 74, characterized in that the viral capsid is selected from the group comprising plll protein, pVIII major capsid protein, Soe, Hoc, gpD, pv1, and variants thereof. 76. Fusion polypeptide according to claim 75, which further comprises a dimerization domain between the variable domain and the viral capsid protein. 77. Fusion polypeptide according to claim 74, further comprising a labeled peptide. 78. Fusing polypeptide according to claim 77, characterized in that the labeled peptide is selected from a group consisting of gD, c-myc, poly-his, a fluorescent protein eB-galactosidase. 79. COMPOSITION containing a polypeptide of one of claims 1 to 78 and a vehicle. 80. POLINUCLEOTIDE, encoding a polypeptide of one of claims 1 to 78. 81. VECTOR containing the polynucleotide of claim 80. 82. HOST CELL containing the claim vector 81. A method of producing a polypeptide comprising culturing the host cell of claim 82 under conditions suitable for the production of a polypeptide and recovery of the polypeptide or antigen-binding fragment thereof. A polypeptide library comprising a variety of polypeptides of one of claims 1 to 78 and in which the library has at least 1 x 10 4 antibody variable domain sequences. 85. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5 -HI, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is 50 of according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 by the fact that X1 is position 95 according to the Kabat numbering system, in which the amino acids at each of the positions of XI-XI? are selected from S and one of A, C, F, G, I, L, N, P, R, T, W or Y, or not present; X18 is selected from G and A; and X19 is selected from F, L, I, and M. 86. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4-X5 -HI, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acids at each of the positions of X1-X5 are selected from S and one of Y, W, Re F; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5 -T-X6-YADSVKG, characterized by the fact that X1 is 50 according to the Kabat numbering system; wherein the amino acids at each of the positions of X1-X6 are selected from S and one between Y, W, R and F; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19 by the fact that X1 is position 95 according to the Kabat numbering system, in which the amino acids at each of the positions of XI-XI 9 are selected from S and one of Y, W, R and or not present; where X18 is selected from G and A; and wherein X19 is selected from F, L, I, and M. A method according to claim 85 or 86, characterized in that the method comprises: (b) producing a variety of polypeptides comprising: (i) CDKL1 comprising a first conservative consensus sequence or variant thereof containing the substitution in one or more positions compared to the corresponding hypervariable consensus sequence: (ii) CDRL2 comprising a second or variant second consensus sequence containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence; and (iii) CDRL3 comprising an amino acid sequence: wherein X1 is position 91 according to the Kabat numbering system; Q-Q-X1-X2-X3-X4-X5-X6-X7-X8-T wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; where X5 is selected from Y and S, or not present; wherein X6 is selected from Y and S, or not present; wherein X7 is selected from P and L; where X8 is selected from F, L, I, and V. A method according to any one of claims 85 to 86, characterized in that the method comprises: (b) producing a variety of polypeptides comprising: (i) CDKL1 comprising a first consensual sequence or variant thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, (ii) CDRL2 comprising a second or variant second consensus sequence containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence; and (iii) CDRL3 comprising an amino acid sequence: Q-Q-X1-X2-X3-X4-P-X5-T, wherein X1 is position 91 according to the Kabat numbering system; wherein X1 is selected from Y and S; where X2 is selected from Y and S; wherein X3 is selected from Y and S; where X4 is selected from Y and S; where X5 is selected from Y and S. 89. The method of claim 85 or 86, wherein the method comprises: (b) producing a variety of polypeptides comprising: (i) CDRL1 comprising a first consensual or variant consensus sequence thereof containing the substitution in one or more positions compared to the corresponding hypervariable consensus sequence: (ii) CDRL2 comprising a second or variant second consensus sequence containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence; and (iii) CDRL3 comprises an amino acid sequence: Q-Q-F-X1-I-X2-X3-X4-X5-I-H, characterized in that G is position 26 and X1 is 28 according to the Kabat numbering system; wherein the amino acids at each of the positions of X1-X5 are selected from S and one between Y, W, R and F; 90. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4- X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is 50 of according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S. and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X9-X10-X11 -X12-X13-X14-X15-X16 -X17-X18-X19-DY, characterized in that X1 is position 95 according to the Kabat numbering system, in which the amino acids at each of the positions of X1-X6 are selected from an amino acid pool in a molar relationship. -50% Y, 25% S and 25% G, and X18 is selected from G and A, and X19 is selected from I, M, L, and F. 91. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4- X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is 50 of according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-DY , wherein X1 is position 95 according to the Kabat numbering system, characterized in that the amino acids at each position of X1-X6 are selected from an amino acid pool in a molar ratio of 25% Y, 50% S, and 25% R; wherein the amino acids at each of the X7-X17 positions are selected from an amino acid pool at a molar ratio 25% Y, 50% S and 25% R, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F. 92. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4- X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Ye S; X 4 is selected from Y and S; and X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is 50 of according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S. and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16 -X17-X18-X19-DY (SEQ ID NO: 27), where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from a pool. amino acids at a molar ratio of 38% Y, 25% S, and 25% G and 12% R; characterized in that amino acids at each position of X7-X17 are selected from an amino acid pool in a molar ratio of 38% Y, 25% S, and 25% G and 12% R, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F. 93. A method of generating a composition comprising a variety of polypeptides comprising: (a) producing a variety of polypeptides comprising: (i) CDRH1 comprises an amino acid sequence: GF-X1-I-X2-X3-X4- X5-IH, characterized in that G is position 26 and X1 is position 28 according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; and X5 is selected from Y and S. (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5-T-X6-YADSVKG, characterized in that X1 is 50 of according to the Kabat numbering system; wherein the amino acid X1 is selected from Y and S; X 2 is selected from Y and S; X3 is selected from Y and S; X4 is selected from Y and S; X5 is selected from Y and S; eX6 is selected from Y and S. and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X9-X10-X11 -X12-X13-X14-X15-X16 -X17-X18-X19-DY (SEQ ID NO: 28), where X1 is position 95 according to the Kabat numbering system, characterized in that amino acids at each of the positions of X1-X6 are selected from a pool. of amino acids in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, -1% E, 1% F, 1% H 1% I, 1% K, 1% L, 1% M, 1% N, -1% P, 1% Q, 1% T, 1% V, and 1% W; characterized in that the amino acids at each position of X7-X17 are selected from an amino acid roll in a molar ratio of 20% Y, 26% S, 26% G, -13% R, 1% A, 1 % D, 1% E, 1% F, 1% H, 1% I, 1% K, -1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W, or not present; where X18 is selected from G and A, and where X19 is selected from I, M, L and F. The method of one of claims 90 to 93, characterized in that the method further comprises: (b) producing a variety of polypeptides comprising: (i) CDRL1 comprises a first consensual or variant consensus sequence thereof containing substitution in one or more positions compared to the corresponding hypervariable consensus sequence, (ii) CDRL2 comprises a second or variant second consensus sequence containing a substitution in one or more positions compared to the corresponding hypervariable consensus sequence, and (iii) CDRL3 comprises the corresponding sequence amino acid QQ-X1-X2-X3-X4-P-X5-T, wherein X1 is position 91 according to the numbering of Kabat and is selected from S and Y; wherein X2 is selected from S and Y; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; where X5 is selected from Y and S. 95. The method of any of claims 87 to 89 or 94, characterized in that the first hypervariable consensus sequence comprises a CDRL1 Kabat consensus sequence. 96. METHOD according to claim 95, characterized in that the first hypervariable consensus sequence is R-A-S-Q-D-V-N-T-A-V-A (SEQ ID NO: 29). The method of one of claims 87 to 89 or 94, characterized in that the second hypervariable consensus sequence comprises a CDRL2 Kabat consensus sequence. 98. The method of claim 97, wherein the second hypervariable consensus sequence is S-A-S-S-L-Y-S (SEQ ID NO: 30). 99. The method of one of claims 85 to 98, characterized in that the variety of polypeptides are encoded by a variety of polynucleotides. A method for the selection of an ANTIGEN-BINDING DOMAIN VARIABLE, which binds to a target antigen from an antibody variable domain library, comprising: (a) interacting the library of claim 84 with the target antigen; separating one or more polypeptides that specifically bind to the target antigen from polypeptides that do not specifically bind to the target antigen by recovering one or more polypeptides that specifically bind to the target antigen and incubating one or more polypeptides that specifically bind to the target antigen in a series of breakdowns consisting of reducing the amount of target antigen to a concentration of from about 0.1 nM to about 1000 nN, and (c) selecting one or more polypeptides that specifically bind to the target antigen and which may bind to a low target antigen concentration. or have an affinity of about 0.1nM to 200nM. Method according to claim 100, characterized in that the antigen is HER2 or DR5. 102. The method of claim 100, wherein the concentration of the target antigen is about 100 to 250 nM. 103. The method of claim 100, wherein the target antigen concentration is about 25 to 100 nM. A method for selecting polypeptides, which select antigen targets from a polypeptide library, comprising: (a) isolating one or more polypeptides that specifically bind to the target antigen by interacting with a library containing a variety of polypeptides of one of the claims from 1 to 79 with a target antigen immobilized under conditions suitable for binding, (b) separating one or more polypeptides that specifically bind to the target antigen from polypeptides not specifically binding to the target antigen, and recovering one or more polypeptides that specifically bind to the target antigen. target antigen to obtain an enriched subpopulation for one or more polypeptides that specifically bind to the target antigen; and (c) optionally repeating steps (a) - (b) at least twice, each repeating using the enriched subpopulation for one or more polypeptides that specifically bind to the target antigen obtained from the last round of selection. 105. The method of claim 104 further comprising: (d) incubating the subpopulation with a labeled target antigen concentration within a range of about 0.1 nM to 1000 nN to form a mixture under conditions suitable for binding; (e) interacting the mixture with an immobilized agent that binds labeled target antigen, (f) detecting one or more polypeptides that specifically bind to the labeled target antigen, and recovering one or more polypeptides that specifically binds to the labeled antigen target antigen. marked target; and (g) optionally repeating steps (d) to (f) for at least two times, each repetition using the enriched subpopulation for one or more polypeptides that specifically bind to the target antigen obtained from the last round of selection using lower target antigen concentration marked than in the previous selection cycle. 106. The method of claim 105, further comprising adding an excess of unlabeled target antigen to the mixture and incubating the mixture for a period of time sufficient to recover one or more polypeptides that specifically bind to the low affinity target antigen. A method for isolating one or more polypeptides, which specifically bind to a high affinity target antigen, comprising: (a) Interacting a library consisting of a polypeptide variety of one of claims 1 to 79 with a target antigen concentration of at least 0.1 nM to 1000 nN to isolate one or more polypeptides that specifically bind to the target antigen, (b) recover one or more polypeptides that specifically bind to the target antigen to obtain enriched subpopulation for a or more polypeptides that specifically bind to the target antigen; and (c) optionally repeating steps (a) and (b) at least twice, each repeating using the subpopulation obtained from the last step and using a decreasing target antigen concentration than used in the previous step to isolate one or more polypeptides. which specifically bind target antigen to a lower concentration of target antigen. 108. A method for treating cancer in a mammal comprising administering a polypeptide of one of claims 1 to 79 to mammals. 109. The method of claim 108, wherein the cancer is selected from breast cancer, cancer colorectal, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), kidney cancer, prostate cancer, cancer. liver disease, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. 110. The method of claim 108 or 109, wherein the antibody is an anti-HER2 antibody. 111. The method of claim 108 or 109, wherein the antibody is an anti-DR5 antibody. Method according to one of Claims 108 to 111, characterized in that the treatment includes the additional step of administering a second therapeutic agent simultaneously or subsequently with the antibody. 113. Method according to claim 112, characterized in that the second therapeutic agent is selected from an antiangiogenic, antineoplastic agent, chemotherapeutic agent and cytotoxic agent. 114. The method of claim 113, wherein the antiangiogenic agent is an anti-hVEGF antibody. 115. A method for treating, a mammal suffering or at risk of developing an inflammatory or autoimmune disease, comprising the step of treating the mammal with an antibody of one of claims 1 to 17. 116. The method of claim 115, wherein the inflammatory or autoimmune disease is arthritereumatoid. 117. Method according to claim 115 or 116, characterized in that the antibody is an anti-DR5 antibody.